Methods and compositions for prevention or treatment of RSV infection

ABSTRACT

Methods and compositions are provided for the prevention or treatment of RSV infection in a human. The methods include administering one or more doses of a composition comprising an siRNA. The dose can be formulated for topical or parenteral administration. Topical administration includes administration as a nasal spray, or by inhalation of respirable particles or droplets. The siRNA comprises a sense strand of ALN-RSV01 and an antisense strand of ALN-RSV01.

STATEMENT OF RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/335,467, filed Dec. 15, 2008, which claims the benefit of U.S.Provisional Application No. 61/013,428, filed Dec. 13, 2007; U.S.Provisional Application No. 61/014,887, filed Dec. 19, 2007; U.S.Provisional Application No. 61/021,309, filed Jan. 15, 2008; U.S.Provisional Application No. 61/034,084, filed Mar. 5, 2008; and U.S.Provisional Application No. 61/049,076, filed Apr. 30, 2008, thedisclosure of each of which is hereby incorporated by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named 19854_Sequence_listing.txt, created on Jan. 20, 2012,with a size of 99,351 bytes. The sequence listing is incorporated byreference.

BACKGROUND

By virtue of its natural function the respiratory tract is exposed to aslew of airborne pathogens that cause a variety of respiratory ailments.Viral infection of the respiratory tract is the most common cause ofinfantile hospitalization in the developed world with an estimated91,000 annual admissions in the US at a cost of $300 M. Humanrespiratory syncytial virus (RSV) and parainfluenza virus (PIV) are twomajor agents of respiratory illness; together, they infect the upper andlower respiratory tracts, leading to croup, pneumonia and bronchiolitis(Openshaw, P. J. M. Respir. Res. 3 (Suppl 1), S15-S20 (2002), Easton, A.J., et al., Clin. Microbiol. Rev. 17, 390-412 (2004)).

RSV alone infects up to 65% of all babies within the first year of life,and essentially all within the first 2 years. It is a significant causeof morbidity and mortality in the elderly as well. Immunity after RSVinfection is neither complete nor lasting, and therefore, repeatedinfections occur in all age groups. Infants experiencing RSVbronchiolitis are more likely to develop wheezing and asthma later inlife. Research for effective treatment and vaccine against RSV has beenongoing for nearly four decades with few successes (Openshaw, P. J. M.Respir. Res. 3 (Suppl 1), S15-S20 (2002), Maggon, K. et al, Rev. Med.Virol. 14, 149-168 (2004)).

Currently, no vaccine is clinically approved for RSV. Strains of RSValso exist for nonhuman animals such as the cattle, goat, pig and sheep,causing loss to agriculture and the dairy and meat industry (Easton, A.J., et al., Clin. Microbiol. Rev. 17, 390-412 (2004)).

Both RSV and PIV contain nonsegmented negative-strand RNA genomes andbelong to the Paramyxoviridae family. A number of features of theseviruses have contributed to the difficulties of prevention and therapy.The viral genomes mutate at a high rate due to the lack of areplicational proof-reading mechanism of the RNA genomes, presenting asignificant challenge in designing a reliable vaccine or antiviral(Sullender, W. M. Clin. Microbiol. Rev. 13, 1-15 (2000)). Promisinginhibitors of the RSV fusion protein (F) were abandoned partly becausethe virus developed resistant mutations that were mapped to the F gene(Razinkov, V., et. al., Antivir. Res. 55, 189-200 (2002), Morton, C. J.et al. Virology 311, 275-288 (2003)). Both viruses associate withcellular proteins, adding to the difficulty of obtaining cell-free viralmaterial for vaccination (Burke, E., et al., Virology 252, 137-148(1998), Burke, E., et al., J. Virol. 74, 669-675 (2000), Gupta, S., etal., J. Virol. 72, 2655-2662 (1998)). Finally, the immunology of both,and especially that of RSV, is exquisitely complex (Peebles, R. S., Jr.,et al., Viral. Immunol. 16, 25-34 (2003), Haynes, L. M., et al., J.Virol. 77, 9831-9844 (2003)). Use of denatured RSV proteins as vaccinesleads to “immunopotentiation” or vaccine-enhanced disease (Polack, F. P.et al. J. Exp. Med. 196, 859-865 (2002)). The overall problem isunderscored by the recent closure of a number of anti-RSV biopharmaprograms.

The RSV genome comprises a single strand of negative sense RNA that is15,222 nucleotides in length and yields eleven major proteins. (Falsey,A. R., and E. E. Walsh, 2000, Clinical Microbiological Reviews13:371-84.) Two of these proteins, the F (fusion) and G (attachment)glycoproteins, are the major surface proteins and the most important forinducing protective immunity. The SH (small hydrophobic) protein, the M(matrix) protein, and the M2 (22 kDa) protein are associated with theviral envelope but do not induce a protective immune response. The N(major nucleocapsid associated protein), P (phosphoprotein), and L(major polymerase protein) proteins are found associated with virionRNA. The two non-structural proteins, NS1 and NS2, presumablyparticipate in host-virus interaction but are not present in infectiousvirions.

Human RSV strains have been classified into two major groups, A and B.The G glycoprotein has been shown to be the most divergent among RSVproteins. Variability of the RSV G glycoprotein between and within thetwo RSV groups is believed to be important to the ability of RSV tocause yearly outbreaks of disease. The G glycoprotein comprises 289-299amino acids (depending on RSV strain), and has an intracellular,transmembrane, and highly glycosylated stalk structure of 90 kDa, aswell as heparin-binding domains. The glycoprotein exists in secreted andmembrane-bound forms.

Successful methods of treating RSV infection are currently unavailable(Maggon K and S. Barik, 2004, Reviews in Medical Virology 14:149-68).Infection of the lower respiratory tract with RSV is a self-limitingcondition in most cases. No definitive guidelines or criteria exist onhow to treat or when to admit or discharge infants and children with thedisease. Hypoxia, which can occur in association with RSV infection, canbe treated with oxygen via a nasal cannula. Mechanical ventilation forchildren with respiratory failure, shock, or recurrent apnea can lowermortality. Some physicians prescribe steroids. However, several studieshave shown that steroid therapy does not affect the clinical course ofinfants and children admitted to the hospital with bronchiolitis. Thuscorticosteroids, alone or in combination with bronchodilators, may beuseless in the management of bronchiolitis in otherwise healthyunventilated patients. In infants and children with underlyingcardiopulmonary diseases, such as bronchopulmonary dysphasia and asthma,steroids have also been used.

Ribavirin, a guanosine analogue with antiviral activity, has been usedto treat infants and children with RSV bronchiolitis since the mid1980s, but many studies evaluating its use have shown conflictingresults. In most centers, the use of ribavirin is now restricted toimmunocompromised patients and to those who are severely ill.

The severity of RSV bronchiolitis has been associated with low serumretinol concentrations, but trials in hospitalized children with RSVbronchiolitis have shown that vitamin A supplementation provides nobeneficial effect. Therapeutic trials of 1500 mg/kg intravenous RSVimmune globulin or 100 mg/kg inhaled immune globulin for RSVlower-respiratory-tract infection have also failed to show substantialbeneficial effects.

In developed countries, the treatment of RSV lower-respiratory-tractinfection is generally limited to symptomatic therapy. Antiviral therapyis usually limited to life-threatening situations due to its high costand to the lack of consensus on efficacy. In developing countries,oxygen is the main therapy (when available), and the only way to lowermortality is through prevention.

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.,Nature 391:806-811, 1998). Short dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function. RNAi has beensuggested as a method of developing a new class of therapeutic agents.However, to date, these have remained mostly as suggestions with nodemonstrate proof that RNAi can be used therapeutically.

Therefore, there is a need for safe and effective vaccines against RSV,especially for infants and children. There is also a need fortherapeutic agents and methods for treating RSV infection at all agesand in immuno-compromised individuals. There is also a need forscientific methods to characterize the protective immune response to RSVso that the pathogenesis of the disease can be studied, and screeningfor therapeutic agents and vaccines can be facilitated. The presentinvention overcomes previous shortcomings in the art by providingmethods and compositions effective for modulating or preventing RSVinfection. Specifically, the present invention advances the art byproviding iRNA agents that have been shown to reduce RSV levels in vitroand in vivo, as well as being effective against both major subtypes ofRSV, and a showing of therapeutic activity of this class of molecules.

SUMMARY

The present invention is based on the in vitro and in vivo demonstrationthat RSV can be inhibited through intranasal administration of iRNAagents, as well as by parenteral administration of such agents, and theidentification of potent iRNA agents from the P, N and L gene of RSVthat can reduce RNA levels with both the A and B subtype of RSV. Basedon these findings, the present invention provides specific compositionsand methods that are useful in reducing RSV mRNA levels, RSV proteinlevels and RSV viral titers in a subject, e.g., a mammal, such as ahuman. It is shown herein that administration of multiple doses of ansiRNA agent over a course of days can provide improved results. E.g., ina preferred embodiment a preselected amount of siRNA agent results inbetter inhibition of gene expression when administered as fractionaldoses over the course of more than one day.

In one aspect, the invention provides for an siRNA composition thatcomprises a therapeutically effective amount of ALN-RSV01. ALN-RSV01 isan siRNA agent with the sense strand sequence (5′ to 3′)GGCUCUUAGCAAAGUCAAGdTdT (SEQ ID NO: 1) and the antisense strand sequence(5′ to 3′) CUUGACUUUGCUAAGAGCCdTdT (SEQ ID NO: 2). ALN-RSV01 is the sameas AL-DP-2017, and the terms are used herein interchangeably. Thestructure of AL-DP-2017 (i.e., ALN-RSV01) and details about itsmanufacture are fully described in co-owned U.S. Provisional ApplicationNo. 61/021,309 filed on Jan. 15, 2008, which is herein incorporated byreference in its entirety, for all purposes.

In one embodiment the invention provides for a lyophilized powder. Inanother embodiment the invention provides for a liquid solution, and inanother embodiment a liquid suspension, and in another embodiment a drypowder comprising said amount of ALN-RSV01. In one embodiment, thetherapeutically effective amount of ALN-RSV01 is less than or equal to150 mg of anhydrous oligonucleotide. In another embodiment, thetherapeutically effective amount is equal to 150 mg of anhydrousoligonucleotide. In another embodiment, the therapeutically effectiveamount is equal to 75 mg of anhydrous oligonucleotide. In oneembodiment, administration of the therapeutically effective amount to ahuman subject produces in the subject no significant increase in thesubject's white cell count. In another embodiment, administration of thetherapeutically effective amount to a human subject produces in theconcentration in a subject's inflammatory cytokine(s). In one embodimentthose cytokine(s) are one or more of CRP, G-CSF, IL1-RA, or TNF.

In one related embodiment, the liquid solution is formulated to have anosmolality ranging from 200-400 mOsm/kg. In certain embodiments, theliquid solution is a buffered. In certain embodiments, the pH of theliquid solution is between 5 and 8. In other embodiments, the pH of theliquid solution is between 5.6 and 7.6. In related embodiments, theliquid solution comprises a sodium phosphate buffer. In still otherembodiments, the concentration of the buffer is between 10 and 100 mM,between 20 and 80 mM, between 30 and 70 mM, between 40 and 60 mM, orequal to or about 50 mM. In yet another embodiment, the pH of thebuffered solution is 6.6.

In one embodiment of the invention, the administration of thetherapeutically effective amount of ALN-RSV01 to a human subjectproduces in said subject no significant increase in a white cell count.In another embodiment, the administration of said therapeuticallyeffective amount to a human subject produces in said subject nosignificant elevation in a CRP concentration, a G-CSF concentration, anIL1-RA concentration or a TNF concentration. In a related embodiment,the siRNA composition further comprises a modification for protectionfrom an exonuclease. In another embodiment, the modification of thesiRNA composition is selected from the group consisting of aphosphorothioate and a hydroxy pyrollidine (hp) linker.

In another embodiment, the therapeutic ALN-RSV01 composition isadministered topically. In related embodiments, the topicaladministration is intranasal or intrapulmonary, e.g., administrationoccurs by inhalation of said composition. In still other relatedembodiments, the patient administers the composition to himself orherself, or a third party (e.g., a guardian or a healthcare practitionersuch as a doctor) can administer the composition to the patient. Incertain embodiments, the composition is administered as an aerosolizedliquid, e.g., a nasal spray. The nasal spray can be administered aBecton-Dickinson Accuspray™ nasal spray system or an equivalent thereof.In related embodiments, the aerosolized liquid is produced by anebulizer. In still other related embodiments, 0.1 ml to 0.6 ml (e.g.,0.1, 0.2, 0.3, 0.4, 0.5, or 0.6 ml) of the aerosolized liquid comprisingALN-RSV01 is administered to each nostril. A plurality of doses can beadministered daily, where a plurality includes two, three, four, or fivedoses. In related embodiments, the administering of the plurality ofdoses reduces RSV protein, mRNA, or titer in a cell of the respiratorytract of said human to at least the same level as an administering of asingle dose that equals the dose provided by said plurality of doses. Inyet other related embodiments, the administering of said plurality ofdoses by inhalation delivers a total dose of between 0.1 and 0.6 mg/kgof anhydrous oligonucleotide to said human.

In certain embodiments, the first of said plurality of doses isadministered before the human patient is infected with RSV (e.g.,prophylactically).

In yet another embodiment, the invention provides a method ofidentifying a compound that is effective in preventing or treating RSVinfection comprising: providing a first human infected with RSV strainMemphis-37 wherein said first human exhibits a reduction in lungfunction during progression of RSV infection; administering to a saidfirst human a test compound, wherein said administering occurs eitherbefore or after RSV infection; assessing progression of the RSVinfection in said first human; comparing progression of RSV infection inthe first human to progression of RSV infection in at least one secondhuman that has not been administered said test compound; identifying thetest compound as a compound that is effective in preventing or treatingRSV infection if progression in the first human subject is reducedcompared to progression in the second human. In a related embodiment,the method further comprises recording or reporting said identificationof the test compound. In yet another related embodiment, the screeningmethod comprises administering the test compound to a third human ifprogression in the first human subject is reduced compared toprogression in the second human.

In one embodiment of the invention, a method is described for preventingor treating a Respiratory Syncytial Virus (RSV) infection in a humanlung transplant recipient, including administering to the human lungtransplant recipient a composition including a therapeutically effectiveamount of ALN-RSV01. In one aspect, the composition is administeredtopically. In a related aspect, the topical administration is intranasalor intrapulmonary. In another related aspect, the topical administrationis intranasal. In another related aspect, the topical administration isintrapulmonary. In a related aspect, the intrapulmonary administrationis by inhalation of the composition. In another aspect, the compositionis administered as an aerosol. In a related aspect, the aerosol is anasal spray. In another related aspect, the aerosol is produced by anebulizer. In a related aspect, the nebulizer is a PARI EFLOW® 30Lnebulizer.

In another embodiment, the method includes administering a plurality ofdoses of the composition. In one aspect, one of the plurality of dosesis administered daily. In another aspect, the plurality of doses is twoor three doses. In another related aspect, the plurality of doses isthree doses.

In another embodiment, the human lung transplant recipient is presentlyinfected with RSV. In one aspect, the human lung transplant recipient ispresently infected with RSV when the first of the plurality of doses isadministered. In a related aspect, the administering reduces RSVprotein, RSV mRNA, RSV peak viral load, time to peak RSV viral load,duration of RSV viral shedding, RSV viral AUC, or RSV titer in a cell ofthe respiratory tract of the human lung transplant recipient.

In another embodiment, the administering of the plurality of dosesreduces RSV protein, RSV mRNA, RSV peak viral load, time to peak RSVviral load, duration of RSV viral shedding, RSV viral AUC, or RSV titerin a cell of the respiratory tract of the human lung transplantrecipient to at least the same level as an administering of a singledose that equals the dose provided by the plurality of doses. In oneaspect, the administering of the plurality of doses is by inhalation anddelivers a total dose of between 0.1 and 0.6 mg/kg of anhydrousoligonucleotide to the human lung transplant recipient.

In another embodiment, the method further includes determining thecharacteristics of RSV infection. In one aspect, the characteristics ofRSV infection are determined by quantitative RT-PCR (qRT-PCR) analysisof a nasal swab sample and/or a sputum sample from the human lungtransplant recipient. In a related aspect, the qRT-PCR is used todetermine RSV mRNA, RSV peak viral load, time to peak RSV viral load,duration of RSV viral shedding, RSV viral AUC, or RSV titer.

In another embodiment, the human lung transplant recipient is receivingbronchodilator therapy. In a related aspect, the human lung transplantrecipient is administered the composition within one hour of receivingbronchodilator therapy.

In another embodiment, the human lung transplant recipient is receivingstandard of care treatment. In a related aspect, the standard of caretreatment includes administration of ribavirin. In another aspect, thehuman lung transplant recipient is administered the composition one totwo hours before administration of ribavirin.

In another embodiment, the administration of the composition to thehuman lung transplant recipient is started within seven days of onset ofsigns and/or symptoms of RSV infection. In one aspect, the signs and/orsymptoms include a decrease in FEV1, fever, new onset rhinorrhea, sorethroat, nasal congestion, cough, wheezing, headache, myalgia, chills, orshortness of breath.

In another embodiment, the aerosolized ALN-RSV01 is administered at aconcentration of 0.6 mg/kg to the human lung transplant recipient byinhalation of the aerosolized ALN-RSV01 once daily for three days usinga PARI EFLOW® 30L nebulizer.

In another embodiment, a method is described for reducing the risk of amedical condition in a human lung transplant recipient includingpreventing or treating a Respiratory Syncytial Virus (RSV) infection ina human lung transplant recipient by administering to the human lungtransplant recipient a composition including a therapeutically effectiveamount of ALN-RSV01, wherein the medical condition is selected from thegroup consisting of acute rejection of transplanted lung, bronchilolitisobliterans syndrome, incidence of intubation, incidence of viral,bacterial, or fungal respiratory infection, and irreversible decline inlung function.

In another aspect, the invention provides a method of preventing ortreating RSV infection by administering a composition that comprises atherapeutically effective amount of ALN-RSV01 to a human patient with arecurring cough that has lasted at least two days. In a relatedembodiment, the method of administrating ALN-RSV01 further comprising astep of diagnosing the human as suffering from a recurring cough lastingat least two days, wherein said diagnosis occurs prior to theadministration of said composition and the human has a recurring coughthat has lasted at least two days. In a related embodiment, the human isless than 18 years old, less than 2 years old, less than 6 months old,or less than 3 months old. In another related embodiment, the patienthas a weakened immune system. In yet another related embodiment, thepatient is dehydrated or at risk of becoming dehydrated. In stillanother embodiment, the patient is administered a dose of Synagis™within 6 weeks of the administration of said composition.

In another aspect, the administration of a therapeutic dose of ALN-RSV01takes less then 10 minutes, or more preferably five minutes or less.

In another aspect, the invention provides a method of preventing ortreating RSV infection by administering a composition that comprises atherapeutically effective amount of ALN-RSV01 to a human patient,further comprising measuring ALN-RSV01-directed cleavage of RSV N genemRNA in said human. In a related embodiment, the ALN-RSV01-directedcleavage is detected by PCR. In another related embodiment, the RSV Ngene mRNA cleavage products are detected in nasal samples of said human.In yet another related embodiment, the RNAi-mediated cleavage ismeasured less than 5 days, or less than 3 days, after administering adose of said composition. In yet another embodiment, the method furthercomprises a step of measuring the titer of RSV in said human.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thisdescription, the drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS AND TABLES

FIG. 1: In vitro inhibition of RSV using iRNA agents. iRNA agentsprovided in Table 1 (a-c) were tested for anti-RSV activity in a plaqueformation assay as described in the Examples. Each column (bar)represents an iRNA agent provided in Table 1 (a-c), e.g., column 1 isthe first agent in Table 1a, etc. Active iRNA agents were identified.

FIG. 2: In vitro dose response inhibition of RSV using iRNA agents.Examples of active agents from Table 1 were tested for anti-RSV activityin a plaque formation assay as described in the Examples at fourconcentrations. A dose dependent-response was found with active iRNAagents tested.

FIG. 3: In vitro inhibition of RSV B subtype using iRNA agents. iRNAagents provided in FIG. 2 were tested for anti-RSV activity againstsubtype B in a plaque formation assay as described in the Examples.Subtype B was inhibited by the iRNA agents tested.

FIG. 4: In vivo inhibition of RSV using iRNA agents. Agents as describedin the figure were tested for anti-RSV activity in a mouse model asdescribed in the Examples. The iRNA agents were effective at reducingviral titers in vivo.

FIG. 5: In vivo inhibition of RSV using AL-DP-1730. AL-DP-1730 wastested for dose-dependent activity using the methods provided in theExamples. The agent showed a dose-dependent response.

FIG. 6: In vivo inhibition of RSV using iRNA agents. iRNA agentsdescribed in the Figure were tested for anti-RSV activity in vivo asdescribed in the Examples.

FIG. 7: In vivo inhibition of RSV using iRNA agents. iRNA agentsdescribed in the Figure were tested for anti-RSV activity in vivo asdescribed in the Examples.

FIG. 8: Sequence analysis of RSV N genes from clinical isolates. FIG. 8discloses SEQ ID NOS 305-306, 306, 306-309, 309-311, 311, 306, 311, 311,309, 311 and 312, respectively, in order of appearance.

FIG. 9: Sequence analysis of RSV N genes from slower growing clinicalRSV isolates showing single base mutation in ALN-RSV01 recognition sitefor isolate LAP6824. FIG. 9 discloses SEQ ID NOS 313, 313, 313-314, 305,304 and 2, respectively, in order of appearance.

FIG. 10: Flow chart illustrating manufacturing process for ALN-RSV01drug substance.

FIG. 11: Illustration of cycle of steps involved in solid-phasesynthesis of ALN-RSV01 drug substance.

FIG. 12: Illustration of cleavage and deprotection reactions followingsolid-phase synthesis of ALN-RSV01 drug substance.

FIG. 13A and FIG. 13B: In vivo inhibition of RSV using iRNA agentsdelivered via aerosol. iRNA agents described in the Figure were testedfor anti-RSV activity in vivo as described in the Example.

FIG. 14: In vivo protection against RSV infection using iRNA agents.iRNA agents described in the Figure were tested prior to RSV challengeto test for protective activity.

FIG. 15: In vitro activity of nebulized iRNA agent. iRNA agent asdescribed was nebulized and shown to retain activity in an in vitroassay of RSV infection.

FIG. 16: Lung function (FEV1 (L)) 30 minutes post-dose in human subjectsafter inhalation of siRNA ALN-RSV01 targeting RSV. Dose in mg/kg(assuming average human weight of 70 kg).

FIG. 17: Mean plasma level of siRNA ALN-RSV01 targeting RSV in man vs.non human primates after inhalation.

FIG. 18: White cell count in human subjects after inhalationmulti-dosing (once daily for three days with total dose of 0.6 mg/kg) ofsiRNA ALN-RSV01 targeting RSV.

FIG. 19: RSV in the lung following administration of siRNA ALN-RSV01.RSV instillation was intranasal (106 pfu). Fixed total dose of siRNA was120 μg. Single administration is indicated by −4 hr, D1, D2, D3; splitdose over three days is indicated by D1+D2+D3.

FIG. 20: Structure of ALN-RSV01 duplex. ALN-RSV01 is a syntheticdouble-stranded RNA (dsRNA) oligonucleotide (SEQ ID NOS 1 and 2) formedby hybridization of two partially complementary single strand RNAs inwhich the 3′ ends are capped with two thymidine units. Hybridizationoccurs across 19 ribonucleotide base pairs to yield the ALN-RSV01molecule. All the phosphodiester functional groups are negativelycharged at neutral pH with a sodium ion as the counter ion.

FIG. 21: In vitro IC50 of ALN-RSV01. Vero cells in 24-well plates weretransfected with decreasing concentration of ALN-RSV01 followed byinfection with 200-300 pfu of RSV/A2. At 5 days post-infection, cellswere fixed, immunostained, and counted. Percent activity against PBS wasplotted and IC50 measured using XLFit software.

FIG. 22: In vivo activity of ALN-RSV01 following single and multidosingin BALB/c mice. A) ALN-RSV01 in vivo dose response curve. BALB/c micewere intranasally treated with ALN-RSV01 at increasing concentrations(25 μg, 50 μg, or 100 μg), control siRNA AL-DP-1730 or PBS 4 hours priorto infection with 1×106 pfu of RSV/A2. Lungs were harvested and virusquantified by standard immunostaining plaque assay and plotted as logpfu/g lung. B) ALN-RSV01 multidose study. BALB/c mice were intranasallytreated with ALN-RSV01 or mismatch siRNA (1730) at either 40 μg, 80 μg,or 120 μg (single dose treatment) or 40 μg (multidose, daily treatment).Lungs were harvested and virus quantified by immunostaining plaque assayon D5. −4, 4 hours prior to infection; D1, day 1 post-infection; D2, day2 post-infection; D3, day 3 post-infection. C) ALN-RSV01 same daymultidose study. BALB/c mice were intranasally treated with ALN-RSV01 ormismatch siRNA (1730) at either 40 μg, 60 μg, 80 μg, or 120 μg forsingle dose groups at days 1 or 2 post-RSV infection, or 40 μg 2× or 3×daily for multidose groups at day 1 or 2 post-RSV infection. Lungs wereharvested and virus quantified by immunostaining plaque assay.

FIG. 23: ALN-RSV01 is a modest stimulatory of IFNα and TNFα in vitro.siRNAs (ALN-RSV01 or mismatch positive controls, 7296 and 5048) weretransfected into peripheral blood mononuclear cells and assayed by ELISAfor induction of cytokines at 24 hrs post transfection. A) IFNαinduction; B) TNFα induction.

FIG. 24: TNFα and IFNα stimulatory mismatched siRNAs do not modulate RSVin vivo. A) siRNAs transfected into peripheral blood mononuclear cellswere assayed for TNFα stimulation at 24 hrs post transfection. B) siRNAstransfected into peripheral blood mononuclear cells were assayed forIFNα stimulation at 24 hrs post transfection. C) Lung viralconcentrations from mice intranasally dosed with RSV at day 0 and withRSV-specific siRNAs (ALN-RSV01) or mismatch control immunostimulatorysiRNAs (2153 and 1730) at 4 hrs pre inoculation. Lung RSV concentrationswere measured by immunostaining plaque assay at day 5 post infection.

FIG. 25: ALN-RSV01 viral inhibition is mediated by RNAi in vivo. Shownis a schematic representation of the 5′-RACE assay used to demonstratethe generation of site-specific cleavage product. Boxed are the resultsof sequence analysis of individual clones isolated from peramplification of linker adapted RSV N gene cDNAs generated from an invivo viral inhibition assay in which mice were inoculated with RSV atday 0 and treated with ALN-RSV01, AL-DP-1730 or PBS at day 3, followedby lung homogenization and evaluation by RACE at day 5 post infection.

FIG. 26: Genotype analysis of RSV primary isolates. Maximum likelihoodphylogenetic trees for (A) RSV A and (B) RSV B based upon analysis ofthe RSV G gene. Bootstrap values >70% (1,000 replicates) are shown atthe corresponding node. Circles indicate isolates analyzed at theALN-RSV01 target site.

FIG. 27: In vitro inhibition of primary RSV isolates by ALN-RSV01. Verocells in 24-well plates were transfected with decreasing concentrationsof ALN-RSV01 followed by infection with 200-300 pfu of RSV primaryisolates. At day 5 post-infection, cells were fixed, immunostained, andcounted. Percent activity against PBS was plotted.

FIG. 28: A Sample Symptom Score Card is depicted.

FIG. 29: Table shows the baseline characteristics of subjects used in arandomized, double-blind, placebo controlled study of a therapeutic iRNA(ALN-RSV01) directed against RSV.

FIG. 30: Table summarizes the primary efficacy outcome: infection rate(cohorts 1-6).

FIG. 31: Table summarizes results of statistical analyses used toevaluate independent effects on infection, where infection is measuredby quantitative culture.

FIG. 32: Table summarizes treatment-emergent adverse events (>5%incidence) documented during course of randomized study of RSV01efficacy.

FIG. 33: Study Design for Randomized Phase II trial of IntranasalALN-RSV01 Versus Placebo in Subjects Experimentally Infected with RSV.i.n.: intranasal; d: study day; h: hours; Rx: dosing with ALN-RSV01 orplacebo; RSV: inoculation with RSV, with quantity of inoculumadministered to subjects in each cohort indicated in the boxes labeled“Cohort 1” and “Cohorts 2-6”. Three subjects (1 active, 2 placebo) werewithdrawn from Cohort 6 due to food-related gastroenteritis havingreceived one study drug dose, and without receiving RSV inoculation.Thus, 88 were evaluated for safety and 85 for efficacy. Subjects werequarantined from Day −2 through Day 11. Nasal washes were obtained dailyduring quarantine except on days −1, 0, and 1 so as not to affect studydrug or RSV inoculation.

FIG. 34: Reverse Kaplan-Meier curves are shown for ALN-RSV01 andplacebo, with infection determined either by (A) quantitative culture(plaque assay) or (B) qRT-PCR. Inoculation with RSV occurred at Day 0,while daily treatment with intranasal ALN-RSV01 or placebo continuedfrom Day −1 through Day 3.

FIG. 35: Quantitative Viral and Disease Measures in Subjects Treatedwith ALNRSV01 or Placebo. Panels A-D: Results shown are for bothinfected and uninfected subjects combined in each treatment arm, withinfection measured either by quantitative culture or qRT-PCR. In panels(A) and (B), the bars show the mean and standard error for viral AUC andpeak viral load over Days 2-11 27 following viral inoculation on Day 0.In panels (C) and (D), the curves show the mean and standard error forviral load on each day from Days 2-11. Panels E-G: Results are for allsubjects in each treatment arm, with individual results (dots) and meanfor the group (horizontal bar) shown for (E) mean total symptom score,(F) mean total directed physical exam (DPE) score, and (G) mean mucusweight. Panels H-I: Mean daily symptom score and standard error forALN-RSV01 or placebo by study day, with results shown for (H) allsubjects or for (I) infected subjects, with infection confirmed byquantitative culture or qRT-PCR. Inoculation with RSV occurred on Day 0,and the period of treatment with either ALN-RSV01 or placebo is shown bythe solid horizontal bar.

FIG. 36. Intranasal Cytokine Concentrations in Subjects Treated withALN-RSV01 or Placebo. Shown are mean daily nasal wash cytokineconcentrations and standard errors on study day −2 and study days 2-11for subjects receiving ALN-RSV01 (closed diamonds) or placebo (opensquares). Inoculation with RSV occurred on Day 0, and the period oftreatment with either ALN-RSV01 or placebo is shown by the solidhorizontal bar.

FIG. 37. Specific iRNA mediated cleavage products in human patients. (A)shows that nasal samples taken from patients treated with ALN-RSV01 haveincreased levels of specific cleavage products relative to patientstreated with placebo. (B) shows a plot of RSV titer versus % correctcleavage (left), and the relationship between % correct cleavage versustime after drug dosing (right).

DETAILED DESCRIPTION

The instant specification may refer to one or more of the followingabbreviations whose meanings are defined in Table 2, below.

TABLE 2 List of Abbreviations A Adenosine AAS Atomic AbsorptionSpectroscopy Ado Adenosine AE Adverse Event ALT Alanine aminotransferaseAST Aspartate aminotransferase AUC Area under the concentration-timecurve AX-HPLC Anion Exchange High Performance Liquid Chromatography BMIBody mass index bpm Beats per minute BUN Blood urea nitrogen C CytidineCa Calcium CBC Complete blood cell [count] CDER Center for DrugEvaluation and Research CFR Code of Federal Regulations CFUColony-Forming Units cGMP Current Good Manufacturing Practices ClChloride COPD Chronic Obstructive Pulmonary Disease CPG Controlled PoreGlass CRF Case Report Form CRO Contract Research Organization CRPc-Reactive Protein CTCAE Common Terminology Criteria for Adverse EventsCV Curriculum Vitae Cyd Cytidine Da Daltons DLT Dose-limiting toxicity(ies) DMT Dimethoxytrityl dsRNA Double-stranded ribonucleic acid dTThymidine dThd Thymidine ECG Electrocardiogram EP European PharmacopeiaESI Electrospray Ionization EU Endotoxin Units FDA Food and DrugAdministration FLP Full Length Product FTIR Fourier Transform InfraredSpectroscopy G Gram G Guanosine GC Gas Chromatography GCP Good ClinicalPractice G-CSF Granulocyte colony stimulating factor GMP GoodManufacturing Practices Guo Guanosine HBsAg Hepatitis B Surface AntigenHct Hematocrit HCV Hepatitis C Virus HFIP Hexafluoroisopropanol HgbHemoglobin HIPAA Health Insurance Portability and Accountability Act HIVHuman Immunodeficiency Virus HPLC High Performance Liquid ChromatographyIB Investigator's Brochure ICF Informed Consent Form ICH InternationalConference on Harmonization ICP Inductively Coupled Plasma ICP-MSInductively Coupled Plasma Mass Spectrometry ID Identity IEC IndependentEthics Committee IL-10 Interleukin 10 IL-8 Interleukin 8 IMPDInvestigational Medicinal Product Dossier INR International NormalizedRatio IRB Institutional Review Board IRC Independent Review CommitteeITT Intent to treat K Potassium kg Kilogram LAL Limulus Amebocyte LysateLC-MS Liquid Chromatography-Mass Spectrometry LDH Lactate dehydrogenaseLLOQ Lower Limit of Quantification M Molar MALDI-TOF Matrix AssistedLaser Desorption Ionization - Time of Flight MCH Mean corpuscularhemoglobin MCHC Mean corpuscular hemoglobin concentration MCV Meancorpuscular volume MedDRA Medical Dictionary for Regulatory Activitiesmg Milligram mL Milliliter mM Millimolar mRNA Messenger Ribonucleic AcidMS Mass Spectrometry MTD Maximum tolerated dose MW Molecular Weight NFull Length Oligonucleotide of the Intermediate Single Strands Na SodiumNaCl Sodium chloride ND Not Detected NF National Formulary nm NanometerNMR Nuclear Magnetic Resonance NOAEL No observed adverse effect levelNOEL No observed effect level NT Not Tested OTC Over the counter PBSPhosphate Buffered Solution PE Physical examination PES PolyethersulfonepH Potential of Hydrogen PI Principal Investigator PK PharmacokineticsPP Per protocol ppm Parts Per Million PT Prothrombin Time PTT Partialthromboplastin time PVDF Polyvinylidene Difluoride q.s. QuantitySufficient QC Quality Control RBC Red Blood Cell RH Relative HumidityRISC RNA induced silencing complex RNA Ribonucleic Acid RNAi RNAinterference RRT Relative Retention Time RSV Respiratory Syncytial VirusRTM RSV Transport Media rt-PCR Reverse transcriptase polymerase chainreaction SAE Serious adverse event SAP Statistical Analysis Plan SARSeasonal allergic rhinitis SEC Size Exclusion Chromatography siRNA SmallInterfering Ribonucleic Acid SOP Standard Operating Procedure SP SafetyPopulation TBDMS Tert-butyldimethylsilyl TCID50 Tissue culture infectivedose producing 50% infection TFF Tangential Flow Filtration Tm DuplexHelix to Coil Melting Temperature TNF Tumor necrosis factor U Uridine UFUltrafiltration UK United Kingdom Urd Uridine US United States USPUnited States Pharmacopeia USP/NF United States Pharmacopeia/NationalFormulary UV Ultraviolet w/v Weight by Volume w/w Weight by Weight WBCWhite Blood Cell WHO World Health Organization

For ease of exposition the term “nucleotide” or “ribonucleotide” issometimes used herein in reference to one or more monomeric subunits ofan RNA agent. It will be understood that the usage of the term“ribonucleotide” or “nucleotide” herein can, in the case of a modifiedRNA or nucleotide surrogate, also refer to a modified nucleotide, orsurrogate replacement moiety, as further described below, at one or morepositions.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogate, all of which are described herein or are wellknown in the RNA synthetic art. While numerous modified RNAs andnucleoside surrogates are described, preferred examples include thosewhich have greater resistance to nuclease degradation than do unmodifiedRNAs. Preferred examples include those that have a 2′ sugarmodification, a modification in a single strand overhang, preferably a3′ single strand overhang, or, particularly if single stranded, a5′-modification which includes one or more phosphate groups or one ormore analogs of a phosphate group.

An “iRNA agent” (abbreviation for “interfering RNA agent”) as usedherein, is an RNA agent, which can down-regulate the expression of atarget gene, e.g., RSV. While not wishing to be bound by theory, an iRNAagent may act by one or more of a number of mechanisms, includingpost-transcriptional cleavage of a target mRNA sometimes referred to inthe art as RNAi, or pre-transcriptional or pre-translational mechanisms.An iRNA agent can be a double stranded (ds) iRNA agent.

A “ds iRNA agent” (abbreviation for “double stranded iRNA agent”), asused herein, is an iRNA agent which includes more than one, andpreferably two, strands in which interchain hybridization can form aregion of duplex structure. A “strand” herein refers to a contiguoussequence of nucleotides (including non-naturally occurring or modifiednucleotides). The two or more strands may be, or each form a part of,separate molecules, or they may be covalently interconnected, e.g. by alinker, e.g. a polyethyleneglycol linker, to form but one molecule. Atleast one strand can include a region which is sufficientlycomplementary to a target RNA. Such strand is termed the “antisensestrand”. A second strand comprised in the dsRNA agent which comprises aregion complementary to the antisense strand is termed the “sensestrand”. However, a ds iRNA agent can also be formed from a single RNAmolecule which is, at least partly; self-complementary, forming, e.g., ahairpin or panhandle structure, including a duplex region. In such case,the term “strand” refers to one of the regions of the RNA molecule thatis complementary to another region of the same RNA molecule.

Although, in mammalian cells, long ds iRNA agents can induce theinterferon response which is frequently deleterious, short ds iRNAagents do not trigger the interferon response, at least not to an extentthat is deleterious to the cell and/or host. The iRNA agents of thepresent invention include molecules which are sufficiently short thatthey do not trigger a deleterious interferon response in mammaliancells. Thus, the administration of a composition of an iRNA agent (e.g.,formulated as described herein) to a mammalian cell can be used tosilence expression of an RSV gene while circumventing a deleteriousinterferon response. Molecules that are short enough that they do nottrigger a deleterious interferon response are termed siRNA agents orsiRNAs herein. “siRNA agent” or “siRNA” as used herein, refers to aniRNA agent, e.g., a ds iRNA agent, that is sufficiently short that itdoes not induce a deleterious interferon response in a human cell, e.g.,it has a duplexed region of less than 30 nucleotide pairs.

The isolated iRNA agents described herein, including ds iRNA agents andsiRNA agents, can mediate silencing of a gene, e.g., by RNA degradation.For convenience, such RNA is also referred to herein as the RNA to besilenced. Such a gene is also referred to as a target gene. Preferably,the RNA to be silenced is a gene product of an RSV gene, particularlythe P, N or L gene product.

As used herein, the phrase “mediates RNAi” refers to the ability of anagent to silence, in a sequence specific manner, a target gene.“Silencing a target gene” means the process whereby a cell containingand/or secreting a certain product of the target gene when not incontact with the agent, will contain and/or secret at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product whencontacted with the agent, as compared to a similar cell which has notbeen contacted with the agent. Such product of the target gene can, forexample, be a messenger RNA (mRNA), a protein, or a regulatory element.

In the anti viral uses of the present invention, silencing of a targetgene will result in a reduction in “viral titer” in the cell or in thesubject. As used herein, “reduction in viral titer” refers to a decreasein the number of viable virus produced by a cell or found in an organismundergoing the silencing of a viral target gene. Reduction in thecellular amount of virus produced will preferably lead to a decrease inthe amount of measurable virus produced in the tissues of a subjectundergoing treatment and a reduction in the severity of the symptoms ofthe viral infection. iRNA agents of the present invention are alsoreferred to as “antiviral iRNA agents”.

As used herein, a “RSV gene” refers to any one of the genes identifiedin the RSV virus genome (See Falsey, A. R., and E. E. Walsh, 2000,Clinical Microbiological Reviews 13:371-84). These genes are readilyknown in the art and include the N, P and L genes which are exemplifiedherein.

As used herein, the term “complementary” is used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between a compound of the invention and a target RNAmolecule, e.g. an RSV viral mRNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least 4 nucleotides.

As used herein, an iRNA agent is “sufficiently complementary” to atarget RNA, e.g., a target mRNA (e.g., a target RSV mRNA) if the iRNAagent reduces the production of a protein encoded by the target RNA in acell. The iRNA agent may also be “exactly complementary” to the targetRNA, e.g., the target RNA and the iRNA agent anneal, preferably to forma hybrid made exclusively of Watson-Crick base pairs in the region ofexact complementarity. A “sufficiently complementary” iRNA agent caninclude an internal region (e.g., of at least 10 nucleotides) that isexactly complementary to a target viral RNA. Moreover, in someembodiments, the iRNA agent specifically discriminates asingle-nucleotide difference. In this case, the iRNA agent only mediatesRNAi if exact complementarity is found in the region (e.g., within 7nucleotides of) the single-nucleotide difference. Preferred iRNA agentswill be based on or consist or comprise the sense and antisensesequences provided in the Examples.

As used herein, “essentially identical” when used referring to a firstnucleotide sequence in comparison to a second nucleotide sequence meansthat the first nucleotide sequence is identical to the second nucleotidesequence except for up to one, two or three nucleotide substitutions(e.g. adenosine replaced by uracil).

As used herein, a “subject” refers to a mammalian organism undergoingtreatment for a disorder mediated by viral expression, such as RSVinfection or undergoing treatment prophylactically to prevent viralinfection. The subject can be any mammal, such as a primate, cow, horse,mouse, rat, dog, pig, goat. In the preferred embodiment, the subject isa human.

As used herein, treating RSV infection refers to the amelioration of anybiological or pathological endpoints that 1) is mediated in part by thepresence of the virus in the subject and 2) whose outcome can beaffected by reducing the level of viral gene products present.

Design and Selection of iRNA Agents

The present invention is based on the demonstration of target genesilencing of a respiratory viral gene in vivo following localadministration to the lungs and nasal passage of an iRNA agent eithervia intranasal administration/inhalation or systemically/parenterallyvia injection and the resulting treatment of viral infection. Thepresent invention is further extended to the use of iRNA agents to morethan one respiratory virus and the treatment of both virus infectionswith co-administration of two or more iRNA agents.

Based on these results, the invention specifically provides an iRNAagent that can be used in treating viral infection, particularlyrespiratory viruses and in particular RSV infection, in isolated formand as a pharmaceutical composition described below. Such agents willinclude a sense strand having at least 15 or more contiguous nucleotidesthat are complementary to a viral gene and an antisense strand having atleast 15 or more contiguous nucleotides that are complementary to thesense strand sequence. Particularly useful are iRNA agents that consistof, consist essentially of or comprise a nucleotide sequence from the PN and L gene of RSV as provided in Table 1 (a-c).

The iRNA agents of the present invention are based on and comprise atleast 15 or more contiguous nucleotides from one of the iRNA agentsshown to be active in Table 1 (a-c). In such agents, the agent canconsist of consist essentially of or comprise the entire sequenceprovided in the table or can comprise 15 or more contiguous residuesprovided in Table 1a-c along with additional nucleotides from contiguousregions of the target gene.

An iRNA agent can be rationally designed based on sequence informationand desired characteristics and the information provided in Table 1(a-c). For example, an iRNA agent can be designed according to sequenceof the agents provided in the Tables as well as in view of the entirecoding sequence of the target gene.

Accordingly, the present invention provides iRNA agents comprising asense strand and antisense strand each comprising a sequence of at least15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides which is essentiallyidentical to, as defined above, a portion of a gene from a respiratoryvirus, particularly the P, N or L protein genes of RSV. Exemplified iRNAagents include those that comprise 15 or more contiguous nucleotidesfrom one of the agents provided in Table 1 (a-c).

The antisense strand of an iRNA agent should be equal to or at least,15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50nucleotides in length. It should be equal to or less than 50, 40, or 30,nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to 23,and 19 to 21 nucleotides in length. Exemplified iRNA agents includethose that comprise 15 or more nucleotides from one of the antisensestrands of one of the agents in Table 1 (a-c).

The sense strand of an iRNA agent should be equal to or at least 15, 1617, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50nucleotides in length. It should be equal to or less than 50, 40, or 30nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to 23,and 19 to 21 nucleotides in length. Exemplified iRNA agents includethose that comprise 15 or more nucleotides from one of the sense strandsof one of the agents in Table 1 (a-c).

The double stranded portion of an iRNA agent should be equal to or atleast, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,40, or 50 nucleotide pairs in length. It should be equal to or less than50, 40, or 30 nucleotides pairs in length. Preferred ranges are 15-30,17 to 25, 19 to 23, and 19 to 21 nucleotides pairs in length.

The agents provided in Table 1 (a-c) are 21 nucleotides in length foreach strand. The iRNA agents contain a 19 nucleotide double strandedregion with a 2 nucleotide overhang on each of the 3′ ends of the agent.These agents can be modified as described herein to obtain equivalentagents comprising at least a portion of these sequences (15 or morecontiguous nucleotides) and or modifications to the oligonucleotidebases and linkages.

Generally, the iRNA agents of the instant invention include a region ofsufficient complementarity to the viral gene, e.g. the P, N or L proteinof RSV, and are of sufficient length in terms of nucleotides, that theiRNA agent, or a fragment thereof, can mediate down regulation of thespecific viral gene. The antisense strands of the iRNA agents of thepresent invention are preferably fully complementary to the mRNAsequences of viral gene, as is herein for the P, L or N proteins of RSV.However, it is not necessary that there be perfect complementaritybetween the iRNA agent and the target, but the correspondence must besufficient to enable the iRNA agent, or a cleavage product thereof, todirect sequence specific silencing, e.g., by RNAi cleavage of an RSVmRNA.

Therefore, the iRNA agents of the instant invention include agentscomprising a sense strand and antisense strand each comprising asequence of at least 16, 17 or 18 nucleotides which is essentiallyidentical, as defined below, to one of the sequences of a viral gene,particularly the P, N or L protein of RSV, such as those agent providedin Table 1 (a-c), except that not more than 1, 2 or 3 nucleotides perstrand, respectively, have been substituted by other nucleotides (e.g.adenosine replaced by uracil), while essentially retaining the abilityto inhibit RSV expression in cultured human cells, as defined below.These agents will therefore possess at least 15 or more nucleotidesidentical to one of the sequences of a viral gene, particularly the P, Lor N protein gene of RSV, but 1, 2 or 3 base mismatches with respect toeither the target viral mRNA sequence or between the sense and antisensestrand are introduced. Mismatches to the target viral mRNA sequence,particularly in the antisense strand, are most tolerated in the terminalregions and if present are preferably in a terminal region or regions,e.g., within 6, 5, 4, or 3 nucleotides of a 5′ and/or 3′ terminus, mostpreferably within 6, 5, 4, or 3 nucleotides of the 5′-terminus of thesense strand or the 3′-terminus of the antisense strand. The sensestrand need only be sufficiently complementary with the antisense strandto maintain the overall double stranded character of the molecule.

It is preferred that the sense and antisense strands be chosen such thatthe iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule, such as those exemplified in Table 1 (a-c).Thus, an iRNA agent contains sense and antisense strands, preferablypaired to contain an overhang, e.g., one or two 5′ or 3′ overhangs butpreferably a 3′ overhang of 2-3 nucleotides. Most embodiments will havea 3′ overhang. Preferred siRNA agents will have single-strandedoverhangs, preferably 3′ overhangs, of 1 to 4, or preferably 2 or 3nucleotides, in length, on one or both ends of the iRNA agent. Theoverhangs can be the result of one strand being longer than the other,or the result of two strands of the same length being staggered. 5′-endsare preferably phosphorylated.

Preferred lengths for the duplexed region are between 15 and 30, mostpreferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe siRNA agent range discussed above. Embodiments in which the twostrands of the siRNA agent are linked, e.g., covalently linked are alsoincluded. Hairpin, or other single strand structures which provide therequired double stranded region, and preferably a 3′ overhang are alsowithin the invention.

Evaluation of Candidate iRNA Agents

A candidate iRNA agent can be evaluated for its ability to down regulatetarget gene expression. For example, a candidate iRNA agent can beprovided, and contacted with a cell, e.g., a human cell, that has beeninfected with or will be infected with the virus of interest, e.g., avirus containing the target gene. Alternatively, the cell can betransfected with a construct from which a target viral gene isexpressed, thus preventing the need for a viral infectivity model. Thelevel of target gene expression prior to and following contact with thecandidate iRNA agent can be compared, e.g., on an RNA, protein level orviral titer. If it is determined that the amount of RNA, protein orvirus expressed from the target gene is lower following contact with theiRNA agent, then it can be concluded that the iRNA agent down-regulatestarget gene expression. The level of target viral RNA or viral proteinin the cell or viral titer in a cell or tissue can be determined by anymethod desired. For example, the level of target RNA can be determinedby Northern blot analysis, reverse transcription coupled with polymerasechain reaction (RT-PCR), bDNA analysis, or RNAse protection assay. Thelevel of protein can be determined, for example, by Western blotanalysis or immuno-fluorescence. Viral titer can be detected through aplaque formation assay.

Stability Testing, Modification, and Retesting of iRNA Agents

A candidate iRNA agent can be evaluated with respect to stability, e.g.,its susceptibility to cleavage by an endonuclease or exonuclease, suchas when the iRNA agent is introduced into the body of a subject. Methodscan be employed to identify sites that are susceptible to modification,particularly cleavage, e.g., cleavage by a component found in the bodyof a subject.

When sites susceptible to cleavage are identified, a further iRNA agentcan be designed and/or synthesized wherein the potential cleavage siteis made resistant to cleavage, e.g. by introduction of a 2′-modificationon the site of cleavage, e.g. a 2′-O-methyl group. This further iRNAagent can be retested for stability, and this process may be iterateduntil an iRNA agent is found exhibiting the desired stability.

In Vivo Testing

An iRNA agent identified as being capable of inhibiting viral geneexpression can be tested for functionality in vivo in an animal model(e.g., in a mammal, such as in mouse, rat or primate) as shown in theexamples. For example, the iRNA agent can be administered to an animal,and the iRNA agent evaluated with respect to its biodistribution,stability, and its ability to inhibit viral, e.g., RSV, gene expressionor to reduce viral titer.

The iRNA agent can be administered directly to the target tissue, suchas by injection, or the iRNA agent can be administered to the animalmodel in the same manner that it would be administered to a human. Asshown herein, the agent can be preferably administered intranasally orvia inhalation as a means of preventing or treating viral infection.

The iRNA agent can also be evaluated for its intracellular distribution.The evaluation can include determining whether the iRNA agent was takenup into the cell. The evaluation can also include determining thestability (e.g., the half-life) of the iRNA agent. Evaluation of an iRNAagent in vivo can be facilitated by use of an iRNA agent conjugated to atraceable marker (e.g., a fluorescent marker suchas fluorescein; aradioactive label, such as 35S, 32P, 33P, or 3H; gold particles; orantigen particles for immunohistochemistry) or other suitable detectionmethod.

The iRNA agent can be evaluated with respect to its ability to downregulate viral gene expression. Levels of viral gene expression in vivocan be measured, for example, by in situ hybridization, or by theisolation of RNA from tissue prior to and following exposure to the iRNAagent. Where the animal needs to be sacrificed in order to harvest thetissue, an untreated control animal will serve for comparison. Targetviral mRNA can be detected by any desired method, including but notlimited to RT-PCR, Northern blot, branched-DNA assay, or RNAseprotection assay. Alternatively, or additionally, viral gene expressioncan be monitored by performing Western blot analysis on tissue extractstreated with the iRNA agent or by ELISA. Viral titer can be determinedusing a pfu assy.

iRNA Chemistry

Described herein are isolated iRNA agents, e.g., ds iRNA agents, thatmediate RNAi to inhibit expression of a viral gene, e.g., the P proteinof RSV.

Methods for producing and purifying iRNA agents are well known to thoseof skill in the art of nucleic acid chemistry. In certain embodimentsthe production methods can include solid phase synthesis usingphosphoramidite monomers with commercial nucleic acid synthesizers. See,e.g., “Solid-Phase Synthesis: A Practical Guide”, (Steven A. Kates andFernando Albericio (eds.), Marcel Dekker, Inc., New York, 2000). Incertain embodiments the invention is practiced using processes andreagents for oligonucleotide synthesis and purification as described inco-owned PCT Application No. PCT/US2005/011490 filed Apr. 5, 2005.

RNA agents discussed herein include otherwise unmodified RNA as well asRNA which have been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, preferably as occur naturally in the human body. The art hasreferred to rare or unusual, but naturally occurring, RNAs as modifiedRNAs, see, e.g., Limbach et al., (1994) Nucleic Acids Res. 22:2183-2196. Such rare or unusual RNAs, often termed modified RNAs(apparently because these are typically the result of apost-transcriptional modification) are within the term unmodified RNA,as used herein. Modified RNA as used herein refers to a molecule inwhich one or more of the components of the nucleic acid, namely sugars,bases, and phosphate moieties, are different from that which occurs innature, preferably different from that which occurs in the human body.While they are referred to as modified “RNAs”, they will of course,because of the modification, include molecules which are not RNAs.Nucleoside surrogates are molecules in which the ribophosphate backboneis replaced with a non-ribophosphate construct that allows the bases tothe presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone. Examples of each of the above are discussed herein.

Modifications described herein can be incorporated into anydouble-stranded RNA and RNA-like molecule described herein, e.g., aniRNA agent. It may be desirable to modify one or both of the antisenseand sense strands of an iRNA agent. As nucleic acids are polymers ofsubunits or monomers, many of the modifications described below occur ata position which is repeated within a nucleic acid, e.g., a modificationof a base, or a phosphate moiety, or the non-linking O of a phosphatemoiety. In some cases the modification will occur at all of the subjectpositions in the nucleic acid but in many, and in fact in most, cases itwill not. By way of example, a modification may only occur at a 3′ or 5′terminal position, may only occur in a terminal region, e.g. at aposition on a terminal nucleotide or in the last 2, 3, 4, 5, or 10nucleotides of a strand. A modification may occur in a double strandregion, a single strand region, or in both. E.g., a phosphorothioatemodification at a non-linking O position may only occur at one or bothtermini, may only occur in a terminal regions, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand, or may occur in double strand and single strand regions,particularly at termini. Similarly, a modification may occur on thesense strand, antisense strand, or both. In some cases, the sense andantisense strand will have the same modifications or the same class ofmodifications, but in other cases the sense and antisense strand willhave different modifications, e.g., in some cases it may be desirable tomodify only one strand, e.g. the sense strand.

Two prime objectives for the introduction of modifications into iRNAagents is their stabilization towards degradation in biologicalenvironments and the improvement of pharmacological properties, e.g.,pharmacodynamic properties, which are further discussed below. Othersuitable modifications to a sugar, base, or backbone of an iRNA agentare described in co-owned PCT Application No. PCT/US2004/01193, filedJan. 16, 2004. An iRNA agent can include a non-naturally occurring base,such as the bases described in co-owned PCT Application No.PCT/US2004/011822, filed Apr. 16, 2004. An iRNA agent can include anon-naturally occurring sugar, such as a non-carbohydrate cyclic carriermolecule. Exemplary features of non-naturally occurring sugars for usein iRNA agents are described in co-owned PCT Application No.PCT/US2004/11829 filed Apr. 16, 2003.

An iRNA agent can include an internucleotide linkage (e.g., the chiralphosphorothioate linkage) useful for increasing nuclease resistance. Inaddition, or in the alternative, an iRNA agent can include a ribosemimic for increased nuclease resistance. Exemplary internucleotidelinkages and ribose mimics for increased nuclease resistance aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can include ligand-conjugated monomer subunits andmonomers for oligonucleotide synthesis. Exemplary monomers are describedin co-owned U.S. application Ser. No. 10/916,185, filed on Aug. 10,2004.

An iRNA agent can have a ZXY structure, such as is described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

An iRNA agent can be complexed with an amphipathic moiety. Exemplaryamphipathic moieties for use with iRNA agents are described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

In another embodiment, the iRNA agent can be complexed to a deliveryagent that features a modular complex. The complex can include a carrieragent linked to one or more of (preferably two or more, more preferablyall three of): (a) a condensing agent (e.g., an agent capable ofattracting, e.g., binding, a nucleic acid, e.g., through ionic orelectrostatic interactions); (b) a fusogenic agent (e.g., an agentcapable of fusing and/or being transported through a cell membrane); and(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type. iRNA agents complexed to a delivery agent aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can have non-canonical pairings, such as between the senseand antisense sequences of the iRNA duplex. Exemplary features ofnon-canonical iRNA agents are described in co-owned PCT Application No.PCT/US2004/07070 filed on Mar. 8, 2004.

Enhanced Nuclease Resistance

An iRNA agent, e.g., an iRNA agent that targets RSV, can have enhancedresistance to nucleases.

For increased nuclease resistance and/or binding affinity to the target,an iRNA agent, e.g., the sense and/or antisense strands of the iRNAagent, can include, for example, 2′-modified ribose units and/orphosphorothioate linkages. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE andaminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino). It isnoteworthy that oligonucleotides containing only the methoxyethyl group(MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilitiescomparable to those modified with the robust phosphorothioatemodification.

“Deoxy” modifications include hydrogen (i.e., deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g., NH2; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2; alkylamino,dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroarylamino, or diheteroaryl amino), —NHC(O)R(R=alkyl, cycloalkyl, aryl,aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality.

Preferred substituents are 2′O-methyl (OMe), 2′-methoxyethyl, 2′-OCH3,2′-O-allyl, 2′-C-allyl, and 2′-fluoro (2′F). In one aspect, both 2′OMeand 2′F are used as substituents on an iRNA agent.

One way to increase resistance is to identify cleavage sites and modifysuch sites to inhibit cleavage, as described in co-owned U.S.Application No. 60/559,917, filed on May 4, 2004. For example, thedinucleotides 5′-UA-3′, 5′ UG 3′,5′-CA-3′, 5′ UU-3′, or 5′-CC-3′ canserve as cleavage sites. Enhanced nuclease resistance can therefore beachieved by modifying the 5′ nucleotide, resulting, for example, in atleast one 5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide wherein theuridine is a 2′-modified nucleotide; at least one 5′-uridine-guanine-3′(5′-UG-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide; at least one 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide,wherein the 5′-cytidine is a 2′-modified nucleotide; at least one5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide; or at least one 5′-cytidine-cytidine-3′(5′-CC-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide. The iRNA agent can include at least 2, at least 3, at least4 or at least 5 of such dinucleotides. In certain embodiments, all thepyrimidines of an iRNA agent carry a 2′-modification, and the iRNA agenttherefore has enhanced resistance to endonucleases.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modifications.

The inclusion of furanose sugars in the oligonucleotide backbone canalso decrease endonucleolytic cleavage. An iRNA agent can be furthermodified by including a 3′ cationic group, or by inverting thenucleoside at the 3′-terminus with a 3′-3′ linkage. In anotheralternative, the 3′-terminus can be blocked with an aminoalkyl group,e.g., a 3′ C5-aminoalkyl dT. Other 3′ conjugates can inhibit 3′-5′exonucleolytic cleavage. While not being bound by theory, a 3′conjugate, such as naproxen or ibuprofen, may inhibit exonucleolyticcleavage by sterically blocking the exonuclease from binding to the3′-end of oligonucleotide. Even small alkyl chains, aryl groups, orheterocyclic conjugates or modified sugars (D-ribose, deoxyribose,glucose etc.) can block 3′-5′-exonucleases.

Similarly, 5′ conjugates can inhibit 5′-3′ exonucleolytic cleavage.While not being bound by theory, a 5′ conjugate, such as naproxen oribuprofen, may inhibit exonucleolytic cleavage by sterically blockingthe exonuclease from binding to the 5′-end of oligonucleotide. Evensmall alkyl chains, aryl groups, or heterocyclic conjugates or modifiedsugars (D-ribose, deoxyribose, glucose etc.) can block3′-5′-exonucleases.

An iRNA agent can have increased resistance to nucleases when a duplexediRNA agent includes a single-stranded nucleotide overhang on at leastone end. In preferred embodiments, the nucleotide overhang includes 1 to4, preferably 2 to 3, unpaired nucleotides. In a preferred embodiment,the unpaired nucleotide of the single-stranded overhang that is directlyadjacent to the terminal nucleotide pair contains a purine base, and theterminal nucleotide pair is a G-C pair, or at least two of the last fourcomplementary nucleotide pairs are G-C pairs. In further embodiments,the nucleotide overhang may have 1 or 2 unpaired nucleotides, and in anexemplary embodiment the nucleotide overhang is 5′-GC-3′. In preferredembodiments, the nucleotide overhang is on the 3′-end of the antisensestrand. In one embodiment, the iRNA agent includes the motif 5′-CGC-3′on the 3′-end of the antisense strand, such that a 2-nt overhang5′-GC-3′ is formed.

In one aspect, a hydroxy pyrollidine (hp) linker provides exonucleaseprotection.

Thus, an iRNA agent can include modifications so as to inhibitdegradation, e.g., by nucleases, e.g., endonucleases or exonucleases,found in the body of a subject. These monomers are referred to herein asNRMs, or Nuclease Resistance promoting Monomers, the correspondingmodifications as NRM modifications. In many cases these modificationswill modulate other properties of the iRNA agent as well, e.g., theability to interact with a protein, e.g., a transport protein, e.g.,serum albumin, or a member of the RISC, or the ability of the first andsecond sequences to form a duplex with one another or to form a duplexwith another sequence, e.g., a target molecule.

One or more different NRM modifications can be introduced into an iRNAagent or into a sequence of an iRNA agent. An NRM modification can beused more than once in a sequence or in an iRNA agent.

NRM modifications include some which can be placed only at the terminusand others which can go at any position. Some NRM modifications that caninhibit hybridization are preferably used only in terminal regions, andmore preferably not at the cleavage site or in the cleavage region of asequence which targets a subject sequence or gene, particularly on theantisense strand. They can be used anywhere in a sense strand, providedthat sufficient hybridization between the two strands of the ds iRNAagent is maintained. In some embodiments it is desirable to put the NRMat the cleavage site or in the cleavage region of a sense strand, as itcan minimize off-target silencing.

In most cases, the NRM modifications will be distributed differentlydepending on whether they are comprised on a sense or antisense strand.If on an antisense strand, modifications which interfere with or inhibitendonuclease cleavage should not be inserted in the region which issubject to RISC mediated cleavage, e.g., the cleavage site or thecleavage region (as described in Elbashir et al., 2001, Genes and Dev.15: 188, hereby incorporated by reference). Cleavage of the targetoccurs about in the middle of a 20 or 21 nt antisense strand, or about10 or 11 nucleotides upstream of the first nucleotide on the target mRNAwhich is complementary to the antisense strand. As used herein cleavagesite refers to the nucleotides on either side of the site of cleavage,on the target mRNA or on the iRNA agent strand which hybridizes to it.Cleavage region means the nucleotides within 1, 2, or 3 nucleotides ofthe cleavage site, in either direction.

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position or with 2, 3, 4, or 5 positions of the terminus,of a sequence which targets or a sequence which does not target asequence in the subject.

Tethered Ligands

The properties of an iRNA agent, including its pharmacologicalproperties, can be influenced and tailored, for example, by theintroduction of ligands, e.g., tethered ligands.

A wide variety of entities, e.g., ligands, can be tethered to an iRNAagent, e.g., to the carrier of a ligand-conjugated monomer subunit.Examples are described below in the context of a ligand-conjugatedmonomer subunit but that is only preferred, entities can be coupled atother points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe carrier. In preferred embodiments, the ligand is attached to thecarrier via an intervening tether. The ligand or tethered ligand may bepresent on the ligand-conjugated monomer when the ligand-conjugatedmonomer is incorporated into the growing strand. In some embodiments,the ligand may be incorporated into a “precursor” ligand-conjugatedmonomer subunit after a “precursor” ligand-conjugated monomer subunithas been incorporated into the growing strand. For example, a monomerhaving, e.g., an amino-terminated tether, e.g., TAP-(CH₂)_(n)NH₂ may beincorporated into a growing sense or antisense strand. In a subsequentoperation, i.e., after incorporation of the precursor monomer subunitinto the strand, a ligand having an electrophilic group, e.g., apentafluorophenyl ester or aldehyde group, can subsequently be attachedto the precursor ligand-conjugated monomer by coupling the electrophilicgroup of the ligand with the terminal nucleophilic group of theprecursor ligand-conjugated monomer subunit tether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; nuclease-resistanceconferring moieties; and natural or unusual nucleobases. Generalexamples include lipophilic molecules, lipids, lectins, steroids (e.g.,uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins, carbohydrates (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid), proteins, protein bindingagents, integrin targeting molecules, polycationics, peptides,polyamines, and peptide mimics.

The ligand may be a naturally occurring or recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.Examples of polyamino acids include polyamino acid is a polylysine(PLL), poly L aspartic acid, poly L-glutamic acid, styrene-maleic acidanhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinylether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamidecopolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers,or polyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic moieties, e.g., cationic lipid,cationic porphyrin, quaternary salt of a polyamine, or an alpha helicalpeptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a thyrotropin, melanotropin, surfactant proteinA, Mucin carbohydrate, a glycosylated polyaminoacid, transferrin,bisphosphonate, polyglutamate, polyaspartate, or an RGD peptide or RGDpeptide mimetic.

Ligands can be proteins, e.g., glycoproteins, lipoproteins, e.g., lowdensity lipoprotein (LDL), or albumins, e.g., human serum albumin (HSA),or peptides, e.g., molecules having a specific affinity for a co-ligand,or antibodies e.g., an antibody, that binds to a specified cell typesuch as a cancer cell, endothelial cell, or bone cell. Ligands may alsoinclude hormones and hormone receptors. They can also includenon-peptidic species, such as cofactors, multivalent lactose,multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine,multivalent mannose, or multivalent fucose. The ligand can be, forexample, a lipopolysaccharide, an activator of p38 MAP kinase, or anactivator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). Other molecules that can bind HSA can also beused as ligands. For example, neproxin or aspirin can be used. A lipidor lipid-based ligand can (a) increase resistance to degradation of theconjugate, (b) increase targeting or transport into a target cell orcell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another aspect, the ligand is a moiety, e.g., a vitamin or nutrient,which is taken up by a target cell, e.g., a proliferating cell. Theseare particularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include the B vitamins, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells.

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennapedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

5′-Phosphate Modifications

In preferred embodiments, iRNA agents are 5′ phosphorylated or include aphosphoryl analog at the 5′ prime terminus. 5′-phosphate modificationsof the antisense strand include those which are compatible with RISCmediated gene silencing. Suitable modifications include:5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure. Other suitable 5′-phosphate modifications will be known tothe skilled person.

The sense strand can be modified in order to inactivate the sense strandand prevent formation of an active RISC, thereby potentially reducingoff-target effects. This can be accomplished by a modification whichprevents 5′-phosphorylation of the sense strand, e.g., by modificationwith a 5′-O-methyl ribonucleotide (see Nykänen et al., (2001) ATPrequirements and small interfering RNA structure in the RNA interferencepathway. Cell 107, 309-321.) Other modifications which preventphosphorylation can also be used, e.g., simply substituting the 5′-OH byH rather than O-Me. Alternatively, a large bulky group may be added tothe 5′-phosphate turning it into a phosphodiester linkage.

Delivery of iRNA Agents to Tissues and Cells

Formulation

The iRNA agents described herein can be formulated for administration toa subject, preferably for administration locally to the lungs and nasalpassage (respiratory tissues) via inhalation or intranasaladministration, or parenterally, e.g., via injection.

For ease of exposition, the formulations, compositions, and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions, and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.

A formulated iRNA agent composition can assume a variety of states. Insome examples, the composition is at least partially crystalline,uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20,or 10% water). In another example, the iRNA agent is in an aqueousphase, e.g., in a solution that includes water, this form being thepreferred form for administration via inhalation.

The aqueous phase or the crystalline compositions can be incorporatedinto a delivery vehicle, e.g., a liposome (particularly for the aqueousphase), or a particle (e.g., a microparticle as can be appropriate for acrystalline composition). Generally, the iRNA agent composition isformulated in a manner that is compatible with the intended method ofadministration.

An iRNA agent preparation can be formulated in combination with anotheragent, e.g., another therapeutic agent or an agent that stabilizes aniRNA agent, e.g., a protein that complexes with the iRNA agent to forman iRNP. Still other agents include chelators, e.g., EDTA (e.g., toremove divalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., abroad specificity RNAse inhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA agent preparation includes another iRNAagent, e.g., a second iRNA agent that can mediate RNAi with respect to asecond gene. Still other preparations can include at least three, five,ten, twenty, fifty, or a hundred or more different iRNA species. In someembodiments, the agents are directed to the same virus but differenttarget sequences. In another embodiment, each iRNA agents is directed toa different virus. As demonstrated in the Example, more than one viruscan be inhibited by co-administering two iRNA agents simultaneously, orat closely time intervals, each one directed to one of the viruses beingtreated.

Treatment Methods and Routes of Delivery

A composition that includes an iRNA agent of the present invention,e.g., an iRNA agent that targets RSV, can be delivered to a subject by avariety of routes. The pharmaceutical compositions of the presentinvention may be administered in a number of ways depending upon whetherlocal or systemic treatment is desired and upon the area to be treated.Administration may be topical (including intranasal or intrapulmonary),oral or parenteral. Exemplary routes include inhalation, intravenous,nasal, or oral delivery.

In general, the delivery of the iRNA agents of the present invention isdone to achieve delivery into the subject to the site of infection. Thisobjective can be achieved through either a local (i.e., topical)administration to the lungs or nasal passage, e.g., into the respiratorytissues via inhalation, nebulization or intranasal administration, orvia systemic administration, e.g., parental administration. Parenteraladministration includes intravenous drip, subcutaneous, intraperitonealor intramuscular injection. The preferred means of administering theiRNA agents of the present invention is through direct topicaladministration to the lungs and/or nasal passage by inhalation of anaerosolized liquid such as a nebulized mist or a nasal spray.

An iRNA agent can be incorporated into pharmaceutical compositionssuitable for administration. For example, compositions can include oneor more iRNA agents and a pharmaceutically acceptable carrier. As usedherein the language “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

Formulations for inhalation, intranasal, or parenteral administrationare well known in the art. Such formulations may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives, an example being PBS or Dextrose 5% in water. For intravenoususe, the total concentration of solutes should be controlled to renderthe preparation isotonic.

The active compounds disclosed herein are preferably administered to thelung(s) or nasal passage of a subject by any suitable means. Activecompounds may be administered by administering an aerosol suspension ofrespirable particles comprised of the active compound or activecompounds, which the subject inhales. The active compound can beaerosolized in a variety of forms, such as, but not limited to, drypowder inhalants, metered dose inhalants, or liquid/liquid suspensions.The respirable particles may be liquid or solid. The particles mayoptionally contain other therapeutic ingredients such as amiloride,benzamil or phenamil, with the selected compound included in an amounteffective to inhibit the reabsorption of water from airway mucoussecretions, as described in U.S. Pat. No. 4,501,729.

The particulate pharmaceutical composition may optionally be combinedwith a carrier to aid in dispersion or transport. A suitable carriersuch as a sugar (i.e., dextrose, lactose, sucrose, trehalose, mannitol)may be blended with the active compound or compounds in any suitableratio (e.g., a 1 to 1 ratio by weight).

In one embodiment, an active compound is topically administered byinhalation. As used in this specification, administration by“inhalation” generally refers to the inspiration of particles comprisedof the active compound that are of respirable size, that is, particlesof a size sufficiently small to pass through the mouth or nose andlarynx upon inhalation and into the bronchi and alveoli of the lungs. Ingeneral, particles ranging from about 1 to 10 microns in size (moreparticularly, less than about 5 microns in size) are respirable andsuitable for administration by inhalation.

In another embodiment, an active compound is topically delivered byintranasal administration. As used in this specification, “intranasal”administration refers to administration of a dosage form formulated anddelivered to topically treat the nasal epithelium. Particles or dropletsused for intranasal administration generally have a diameter that islarger than those used for administration by inhalation. For intranasaladministration, a particle size in the range of 10-500 microns ispreferred to ensure retention in the nasal cavity. Particles ofnon-respirable size which are included in the aerosol tend to deposit inthe throat and be swallowed, and the quantity of non-respirableparticles in the aerosol is preferably minimized.

Liquid pharmaceutical compositions of active compound for producing anaerosol can be prepared by combining the active compound with a suitablevehicle, such as sterile pyrogen free water. In certain embodimentshypertonic saline solutions are used to carry out the present invention.These are preferably sterile, pyrogen-free solutions, comprising fromone to fifteen percent (by weight) of a physiologically acceptable salt,and more preferably from three to seven percent by weight of thephysiologically acceptable salt.

Aerosols of liquid particles comprising the active compound may beproduced by any suitable means, such as with a pressure-driven jetnebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No.4,501,729. Nebulizers are commercially available devices which transformsolutions or suspensions of the active ingredient into a therapeuticaerosol mist either by means of acceleration of compressed gas,typically air or oxygen, through a narrow venturi orifice or by means ofultrasonic agitation.

Suitable formulations for use in nebulizers consist of the activeingredient in a liquid carrier, the active ingredient comprising up to40% w/w of the formulation, but preferably less than 20% w/w. Thecarrier is typically water (and most preferably sterile, pyrogen-freewater) or a dilute aqueous alcoholic solution, preferably made isotonic,but may be hypertonic with body fluids by the addition of, for example,sodium chloride. Optional additives include preservatives if theformulation is not made sterile, for example, methyl hydroxybenzoate,antioxidants, flavoring agents, volatile oils, buffering agents andsurfactants.

Aerosols of solid particles comprising the active compound may likewisebe produced with any solid particulate therapeutic aerosol generator.Aerosol generators for administering solid particulate therapeutics to asubject produce particles which are respirable and generate a volume ofaerosol containing a predetermined metered dose of a therapeutic at arate suitable for human administration. One illustrative type of solidparticulate aerosol generator is an insufflator. Suitable formulationsfor administration by insufflation include finely comminuted powderswhich may be delivered by means of an insufflator or taken into thenasal cavity in the manner of a snuff. In the insufflator, the powder(e.g., a metered dose thereof effective to carry out the treatmentsdescribed herein) is contained in capsules or cartridges, typically madeof gelatin or plastic, which are either pierced or opened in situ andthe powder delivered by air drawn through the device upon inhalation orby means of a manually-operated pump. The powder employed in theinsufflator consists either solely of the active ingredient or of apowder blend comprising the active ingredient, a suitable powderdiluent, such as lactose, and an optional surfactant. The activeingredient typically comprises from 0.1 to 100 w/w of the formulation.

A second type of illustrative aerosol generator comprises a metered doseinhaler. Metered dose inhalers are pressurized aerosol dispensers,typically containing a suspension or solution formulation of the activeingredient in a liquefied propellant. During use these devices dischargethe formulation through a valve adapted to deliver a metered volume,typically from 10 μl to 200 μl, to produce a fine particle spraycontaining the active ingredient. Suitable propellants include certainchlorofluorocarbon compounds, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.The formulation may additionally contain one or more co-solvents, forexample, ethanol, surfactants, such as oleic acid or sorbitan trioleate,antioxidant and suitable flavoring agents.

Administration can be provided by the subject or by another person,e.g., a caregiver. A caregiver can be any entity involved with providingcare to the human: for example, a hospital, hospice, doctor's office,outpatient clinic; a healthcare worker such as a doctor, nurse, or otherpractitioner; or a spouse or guardian, such as a parent. The medicationcan be provided in measured doses or in a dispenser which delivers ametered dose.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response. Inone embodiment, therapeutically effective amounts of two or more iRNAagents, each one directed to a different respiratory virus, e.g., RSV,and FIV are administered concurrently to a subject.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carriercan be taken into the lungs with no significant adverse toxicologicaleffects on the lungs.

The term “co-administration” refers to administering to a subject two ormore agents, and in particular two or more iRNA agents. The agents canbe contained in a single pharmaceutical composition and be administeredat the same time, or the agents can be contained in separate formulationand administered serially to a subject. So long as the two agents can bedetected in the subject at the same time, the two agents are said to beco-administered.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-beta-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, threhalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

Dosage. An iRNA agent can be administered at a unit dose less than about75 mg per kg of bodyweight, or less than about 70, 60, 50, 40, 30, 20,10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg ofbodyweight, and less than 200 nmol of iRNA agent (e.g., about 4.4×1016copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15,7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015nmol of iRNA agent per kg of bodyweight. The unit dose, for example, canbe administered by an inhaled dose or nebulization or by injection. Inone example, dosage ranges of 0.02-25 mg/kg is used.

Delivery of an iRNA agent directly to the lungs or nasal passage can beat a dosage on the order of about 1 mg to about 150 mg/nasal passage,such as, e.g., 25, 50, 75, 100 or 150 mg/nasal passage.

The dosage can be an amount effective to treat or prevent a disease ordisorder.

In one embodiment, the unit dose is administered once a day. In otherusage, a unit dose is administered twice the first day and then daily.Alternatively, unit dosing can be less than once a day, e.g., less thanevery 2, 4, 8 or 30 days. In another embodiment, the unit dose is notadministered with a frequency (e.g., not a regular frequency). Forexample, the unit dose may be administered a single time. Because iRNAagent mediated silencing can persist for several days afteradministering the iRNA agent composition, in many instances, it ispossible to administer the composition with a frequency of less thanonce per day, or, for some instances, only once for the entiretherapeutic regimen.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an iRNA agent, e.g., a double-stranded iRNAagent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into an siRNA agent, or a DNA which encodes aniRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, orprecursor thereof). The maintenance dose or doses are generally lowerthan the initial dose, e.g., one-half less of the initial dose. Amaintenance regimen can include treating the subject with a dose ordoses ranging from 0.01 μg to 75 mg/kg of body weight per day, e.g., 70,60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or0.0005 mg per kg of bodyweight per day. The maintenance doses arepreferably administered no more than once every 5-14 days. Further, thetreatment regimen may last for a period of time which will varydepending upon the nature of the particular disease, its severity andthe overall condition of the patient. In preferred embodiments thedosage may be delivered no more than once per day, e.g., no more thanonce per 24, 36, 48, or more hours, e.g., no more than once every 5 or 8days. Following treatment, the patient can be monitored for changes inhis condition and for alleviation of the symptoms of the disease state.The dosage of the compound may either be increased in the event thepatient does not respond significantly to current dosage levels, or thedose may be decreased if an alleviation of the symptoms of the diseasestate is observed, if the disease state has been ablated, or ifundesired side-effects are observed.

In one embodiment, the iRNA agent pharmaceutical composition includes aplurality of iRNA agent species. The iRNA agent species can havesequences that are non-overlapping and non-adjacent with respect to anaturally occurring target sequence, e.g., a target sequence of the RSVgene. In another embodiment, the plurality of iRNA agent species isspecific for different naturally occurring target genes. For example, aniRNA agent that targets the P protein gene of RSV can be present in thesame pharmaceutical composition as an iRNA agent that targets adifferent gene, for example the N protein gene. In another embodiment,the iRNA agents are specific for different viruses, e.g., RSV.

The concentration of the iRNA agent composition is an amount sufficientto be effective in treating or preventing a disorder or to regulate aphysiological condition in humans. The concentration or amount of iRNAagent administered will depend on the parameters determined for theagent and the method of administration, e.g., nasal, buccal, orpulmonary. For example, nasal formulations tend to require much lowerconcentrations of some ingredients in order to avoid irritation orburning of the nasal passages. It is sometimes desirable to dilute anoral formulation up to 10-100 times in order to provide a suitable nasalformulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. It will also be appreciated thatthe effective dosage of an iRNA agent such as an siRNA agent used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays. For example, the subject can be monitoredafter administering an iRNA agent composition. Based on information fromthe monitoring, an additional amount of the iRNA agent composition canbe administered.

Generally speaking, administration of the therapeutic compositions ofthe invention can occur in a hospital or clinical setting (“inpatientmanagement”), or can occur in an “outpatient” setting, e.g., at thepatient's home. Inpatient treatment would be most appropriate where thepatient is suffering from severe symptoms of RSV, including apnea,respiratory distress, low oxygen intake, and/or hydration or starvation.Depending on the patient's age and ability, the patient can administerthe therapeutic compositions to himself or herself. Treatment with thetherapeutic compositions of the invention may be accompanied by, e.g.,hydration, steam, bronchodilators (e.g., albuteraol), anti-fever oranti-inflammatory medications (e.g., Tylenol) and the intake of food,liquids, and/or vitamin supplements. In addition, and typically in aninpatient setting, the patient may also receive oxygen treatment,intravenous hydration, and treatment with steroids.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1 Designing Antiviral siRNAs Against RSV mRNA

siRNA against RSV P, N and L mRNA were synthesized chemically using knowprocedures. The siRNA sequences and some inhibition cross-subtypeactivity and IC50 values as described below are listed in Tables 1a, 1b,and 1c.

TABLE 1a RSV L gene siRNA Actual Whitehead SEQ ID SEQ ID RSV L genestart Start Pos NO. Sense NO. Antisense duplex 3 1 3GGAUCCCAUUAUUAAUGGAdTdT 117 UCCAUUAAUAAUGGGAUCCdTdT AL-DP-2024 4 2 4GAUCCCAUUAUUAAUGGAAdTdT 118 UUCCAUUAAUAAUGGGAUCdTdT AL-DP-2026 49 47 5AGUUAUUUAAAAGGUGUUAdTdT 119 UAACACCUUUUAAAUAACUdTdT AL-DP-2116 50 48 6GUUAUUUAAAAGGUGUUAUdTdT 120 AUAACACCUUUUAAAUAACdTdT AL-DP-2117 53 51 7AUUUAAAAGGUGUUAUCUCdTdT 121 GAGAUAACACCUUUUAAAUdTdT AL-DP-2118 55 53 8UUAAAAGGUGUUAUCUCUUdTdT 122 AAGAGAUAACACCUUUUAAdTdT AL-DP-2119 156 154 9AAGUCCACUACUAGAGCAUdTdT 123 AUGCUCUAGUAGUGGACUUdTdT AL-DP-2027 157 15510 AGUCCACUACUAGAGCAUAdTdT 124 UAUGCUCUAGUAGUGGACUdTdT AL-DP-2028 158156 11 GUCCACUACUAGAGCAUAUdTdT 125 AUAUGCUCUAGUAGUGGACdTdT AL-DP-2029159 157 12 UCCACUACUAGAGCAUAUGdTdT 126 CAUAUGCUCUAGUAGUGGAdTdTAL-DP-2030 341 339 13 GAAGAGCUAUAGAAAUAAGdTdT 127CUUAUUUCUAUAGCUCUUCdTdT AL-DP-2120 344 342 14 GAGCUAUAGAAAUAAGUGAdTdT128 UCACUUAUUUCUAUAGCUCdTdT AL-DP-2121 347 345 15CUAUAGAAAUAAGUGAUGUdTdT 129 ACAUCACUUAUUUCUAUAGdTdT AL-DP-2031 554 55216 UCAAAACAACACUCUUGAAdTdT 130 UUCAAGAGUGUUGUUUUGAdTdT AL-DP-2122 10041002 17 UAGAGGGAUUUAUUAUGUCdTdT 131 GACAUAAUAAAUCCCUCUAdTdT AL-DP-21231408 1406 18 AUAAAAGGGUUUGUAAAUAdTdT 132 UAUUUACAAACCCUUUUAUdTdTAL-DP-2124 1867 1865 19 CUCAGUGUAGGUAGAAUGUdTdT 133ACAUUCUACCUACACUGAGdTdT AL-DP-2032 1868 1866 20 UCAGUGUAGGUAGAAUGUUdTdT134 AACAUUCUACCUACACUGAdTdT AL-DP-2033 1869 1867 21CAGUGUAGGUAGAAUGUUUdTdT 135 AAACAUUCUACCUACACUGdTdT AL-DP-2034 1870 186822 AGUGUAGGUAGAAUGUUUGdTdT 136 CAAACAUUCUACCUACACUdTdT AL-DP-2112 18711869 23 GUGUAGGUAGAAUGUUUGCdTdT 137 GCAAACAUUCUACCUACACdTdT AL-DP-21131978 1976 24 ACAAGAUAUGGUGAUCUAGdTdT 138 CUAGAUCACCAUAUCUUGUdTdTAL-DP-2035 2104 2102 25 AGCAAAUUCAAUCAAGCAUdTdT 139AUGCUUGAUUGAAUUUGCUdTdT AL-DP-2036 2105 2103 26 GCAAAUUCAAUCAAGCAUUdTdT140 AAUGCUUGAUUGAAUUUGCdTdT AL-DP-2037 2290 2288 27GAUGAACAAAGUGGAUUAUdTdT 141 AUAAUCCACUUUGUUCAUCdTdT AL-DP-2038 2384 238228 UAAUAUCUCUCAAAGGGAAdTdT 142 UUCCCUUUGAGAGAUAUUAdTdT AL-DP-2125 23862384 29 AUAUCUCUCAAAGGGAAAUdTdT 143 AUUUCCCUUUGAGAGAUAUdTdT AL-DP-21262387 2385 30 UAUCUCUCAAAGGGAAAUUdTdT 144 AAUUUCCCUUUGAGAGAUAdTdTAL-DP-2127 2485 2483 31 CAUGCUCAAGCAGAUUAUUdTdT 145AAUAAUCUGCUUGAGCAUGdTdT AL-DP-2039 2487 2485 32 UGCUCAAGCAGAUUAUUUGdTdT146 CAAAUAAUCUGCUUGAGCAdTdT AL-DP-2040 2507 2505 33UAGCAUUAAAUAGCCUUAAdTdT 147 UUAAGGCUAUUUAAUGCUAdTdT AL-DP-2041 2508 250634 AGCAUUAAAUAGCCUUAAAdTdT 148 UUUAAGGCUAUUUAAUGCUdTdT AL-DP-2114 25092507 35 GCAUUAAAUAGCCUUAAAUdTdT 149 AUUUAAGGCUAUUUAAUGCdTdT AL-DP-20422510 2508 36 CAUUAAAUAGCCUUAAAUUdTdT 150 AAUUUAAGGCUAUUUAAUGdTdTAL-DP-2043 2765 2763 37 UAUUAUGCAGUUUAAUAUUdTdT 151AAUAUUAAACUGCAUAAUAdTdT AL-DP-2044 2767 2765 38 UUAUGCAGUUUAAUAUUUAdTdT152 UAAAUAUUAAACUGCAUAAdTdT AL-DP-2045 3283 3281 39AAAAGUGCACAACAUUAUAdTdT 153 UAUAAUGUUGUGCACUUUUdTdT AL-DP-2128 3284 328240 AAAGUGCACAACAUUAUACdTdT 154 GUAUAAUGUUGUGCACUUUdTdT AL-DP-2046 33383336 41 AUAUAGAACCUACAUAUCCdTdT 155 GGAUAUGUAGGUUCUAUAUdTdT AL-DP-20473339 3337 42 UAUAGAACCUACAUAUCCUdTdT 156 AGGAUAUGUAGGUUCUAUAdTdTAL-DP-2048 3365 3363 43 UAAGAGUUGUUUAUGAAAGdTdT 157CUUUCAUAAACAACUCUUAdTdT AL-DP-2129 4021 4019 44 ACAGUCAGUAGUAGACCAUdTdT158 AUGGUCUACUACUGACUGUdTdT AL-DP-2049 4022 4020 45CAGUCAGUAGUAGACCAUGdTdT 159 CAUGGUCUACUACUGACUGdTdT AL-DP-2050 4023 402146 AGUCAGUAGUAGACCAUGUdTdT 160 ACAUGGUCUACUACUGACUdTdT AL-DP-2051 40244022 47 GUCAGUAGUAGACCAUGUGdTdT 161 CACAUGGUCUACUACUGACdTdT AL-DP-20524025 4023 48 UCAGUAGUAGACCAUGUGAdTdT 162 UCACAUGGUCUACUACUGAdTdTAL-DP-2053 4037 4035 49 CAUGUGAAUUCCCUGCAUCdTdT 163GAUGCAGGGAAUUCACAUGdTdT AL-DP-2054 4038 4036 50 AUGUGAAUUCCCUGCAUCAdTdT164 UGAUGCAGGGAAUUCACAUdTdT AL-DP-2055 4039 4037 51UGUGAAUUCCCUGCAUCAAdTdT 165 UUGAUGCAGGGAAUUCACAdTdT AL-DP-2056 4040 403852 GUGAAUUCCCUGCAUCAAUdTdT 166 AUUGAUGCAGGGAAUUCACdTdT AL-DP-2115 40434041 53 AAUUCCCUGCAUCAAUACCdTdT 167 GGUAUUGAUGCAGGGAAUUdTdT AL-DP-20574051 4049 54 GCAUCAAUACCAGCUUAUAdTdT 168 UAUAAGCUGGUAUUGAUGCdTdTAL-DP-2058 4052 4050 55 CAUCAAUACCAGCUUAUAGdTdT 169CUAUAAGCUGGUAUUGAUGdTdT AL-DP-2059 4057 4055 56 AUACCAGCUUAUAGAACAAdTdT170 UUGUUCUAUAAGCUGGUAUdTdT AL-DP-2060 4058 4056 57UACCAGCUUAUAGAACAACdTdT 171 GUUGUUCUAUAAGCUGGUAdTdT AL-DP-2061 4059 405758 ACCAGCUUAUAGAACAACAdTdT 172 UGUUGUUCUAUAAGCUGGUdTdT AL-DP-2062 40604058 59 CCAGCUUAUAGAACAACAAdTdT 173 UUGUUGUUCUAUAAGCUGGdTdT AL-DP-20634061 4059 60 CAGCUUAUAGAACAACAAAdTdT 174 UUUGUUGUUCUAUAAGCUGdTdTAL-DP-2064 4067 4065 61 AUAGAACAACAAAUUAUCAdTdT 175UGAUAAUUUGUUGUUCUAUdTdT AL-DP-2065 4112 4110 62 UAUUAACAGAAAAGUAUGGdTdT176 CCAUACUUUUCUGUUAAUAdTdT AL-DP-2130 4251 4249 63UGAGAUACAUUUGAUGAAAdTdT 177 UUUCAUCAAAUGUAUCUCAdTdT AL-DP-2066 4252 425064 GAGAUACAUUUGAUGAAACdTdT 178 GUUUCAUCAAAUGUAUCUCdTdT AL-DP-2067 42544252 65 GAUACAUUUGAUGAAACCUdTdT 179 AGGUUUCAUCAAAUGUAUCdTdT AL-DP-20684255 4253 66 AUACAUUUGAUGAAACCUCdTdT 180 GAGGUUUCAUCAAAUGUAUdTdTAL-DP-2069 4256 4254 67 UACAUUUGAUGAAACCUCCdTdT 181GGAGGUUUCAUCAAAUGUAdTdT AL-DP-2074 4313 4311 68 AAGUGAUACAAAAACAGCAdTdT182 UGCUGUUUUUGUAUCACUUdTdT AL-DP-2131 4314 4312 69AGUGAUACAAAAACAGCAUdTdT 183 AUGCUGUUUUUGUAUCACUdTdT AL-DP-2132 4316 431470 UGAUACAAAAACAGCAUAUdTdT 184 AUAUGCUGUUUUUGUAUCAdTdT AL-DP-2133 44734471 71 UUUAAGUACUAAUUUAGCUdTdT 185 AGCUAAAUUAGUACUUAAAdTdT AL-DP-20754474 4472 72 UUAAGUACUAAUUUAGCUGdTdT 186 CAGCUAAAUUAGUACUUAAdTdTAL-DP-2076 4475 4473 73 UAAGUACUAAUUUAGCUGGdTdT 187CCAGCUAAAUUAGUACUUAdTdT AL-DP-2077 4476 4474 74 AAGUACUAAUUUAGCUGGAdTdT188 UCCAGCUAAAUUAGUACUUdTdT AL-DP-2078 4477 4475 75AGUACUAAUUUAGCUGGACdTdT 189 GUCCAGCUAAAUUAGUACUdTdT AL-DP-2079 4478 447676 GUACUAAUUUAGCUGGACAdTdT 190 UGUCCAGCUAAAUUAGUACdTdT AL-DP-2080 44804478 77 ACUAAUUUAGCUGGACAUUdTdT 191 AAUGUCCAGCUAAAUUAGUdTdT AL-DP-20814483 4481 78 AAUUUAGCUGGACAUUGGAdTdT 192 UCCAAUGUCCAGCUAAAUUdTdTAL-DP-2082 4484 4482 79 AUUUAGCUGGACAUUGGAUdTdT 193AUCCAAUGUCCAGCUAAAUdTdT AL-DP-2083 4486 4484 80 UUAGCUGGACAUUGGAUUCdTdT194 GAAUCCAAUGUCCAGCUAAdTdT AL-DP-2084 4539 4537 81UUUUGAAAAAGAUUGGGGAdTdT 195 UCCCCAAUCUUUUUCAAAAdTdT AL-DP-2134 4540 453882 UUUGAAAAAGAUUGGGGAGdTdT 196 CUCCCCAAUCUUUUUCAAAdTdT AL-DP-2135 45424540 83 UGAAAAAGAUUGGGGAGAGdTdT 197 CUCUCCCCAAUCUUUUUCAdTdT AL-DP-21364543 4541 84 GAAAAAGAUUGGGGAGAGGdTdT 198 CCUCUCCCCAAUCUUUUUCdTdTAL-DP-2137 4671 4669 85 UAUGAACACUUCAGAUCUUdTdT 199AAGAUCUGAAGUGUUCAUAdTdT AL-DP-2085 4672 4670 86 AUGAACACUUCAGAUCUUCdTdT200 GAAGAUCUGAAGUGUUCAUdTdT AL-DP-2086 4867 4865 87UGCCCUUGGGUUGUUAACAdTdT 201 UGUUAACAACCCAAGGGCAdTdT AL-DP-2087 4868 486688 GCCCUUGGGUUGUUAACAUdTdT 202 AUGUUAACAACCCAAGGGCdTdT AL-DP-2088 55445542 89 UAUAGCAUUCAUAGGUGAAdTdT 203 UUCACCUAUGAAUGCUAUAdTdT AL-DP-20895545 5543 90 AUAGCAUUCAUAGGUGAAGdTdT 204 CUUCACCUAUGAAUGCUAUdTdTAL-DP-2090 5546 5544 91 UAGCAUUCAUAGGUGAAGGdTdT 205CCUUCACCUAUGAAUGCUAdTdT AL-DP-2091 5550 5548 92 AUUCAUAGGUGAAGGAGCAdTdT206 UGCUCCUUCACCUAUGAAUdTdT AL-DP-2092 5640 5638 93UUGCAAUGAUCAUAGUUUAdTdT 207 UAAACUAUGAUCAUUGCAAdTdT AL-DP-2093 5641 563994 UGCAAUGAUCAUAGUUUACdTdT 208 GUAAACUAUGAUCAUUGCAdTdT AL-DP-2094 56425640 95 GCAAUGAUCAUAGUUUACCdTdT 209 GGUAAACUAUGAUCAUUGCdTdT AL-DP-20955643 5641 96 CAAUGAUCAUAGUUUACCUdTdT 210 AGGUAAACUAUGAUCAUUGdTdTAL-DP-2096 5644 5642 97 AAUGAUCAUAGUUUACCUAdTdT 211UAGGUAAACUAUGAUCAUUdTdT AL-DP-2097 5645 5643 98 AUGAUCAUAGUUUACCUAUdTdT212 AUAGGUAAACUAUGAUCAUdTdT AL-DP-2098 5647 5645 99GAUCAUAGUUUACCUAUUGdTdT 213 CAAUAGGUAAACUAUGAUCdTdT AL-DP-2138 5648 5646100 AUCAUAGUUUACCUAUUGAdTdT 214 UCAAUAGGUAAACUAUGAUdTdT AL-DP-2139 56495647 101 UCAUAGUUUACCUAUUGAGdTdT 215 CUCAAUAGGUAAACUAUGAdTdT AL-DP-21405650 5648 102 CAUAGUUUACCUAUUGAGUdTdT 216 ACUCAAUAGGUAAACUAUGdTdTAL-DP-2099 5651 5649 103 AUAGUUUACCUAUUGAGUUdTdT 217AACUCAAUAGGUAAACUAUdTdT AL-DP-2100 5752 5750 104 CAUUGGUCUUAUUUACAUAdTdT218 UAUGUAAAUAAGACCAAUGdTdT AL-DP-2101 5754 5752 105UUGGUCUUAUUUACAUAUAdTdT 219 UAUAUGUAAAUAAGACCAAdTdT AL-DP-2102 5755 5753106 UGGUCUUAUUUACAUAUAAdTdT 220 UUAUAUGUAAAUAAGACCAdTdT AL-DP-2103 57565754 107 GGUCUUAUUUACAUAUAAAdTdT 221 UUUAUAUGUAAAUAAGACCdTdT AL-DP-21415919 5917 108 AUAUCAUGCUCAAGAUGAUdTdT 222 AUCAUCUUGAGCAUGAUAUdTdTAL-DP-2142 5920 5918 109 UAUCAUGCUCAAGAUGAUAdTdT 223UAUCAUCUUGAGCAUGAUAdTdT AL-DP-2104 5934 5932 110 UGAUAUUGAUUUCAAAUUAdTdT224 UAAUUUGAAAUCAAUAUCAdTdT AL-DP-2105 6016 6014 111UACUUAGUCCUUACAAUAGdTdT 225 CUAUUGUAAGGACUAAGUAdTdT AL-DP-2106 6019 6017112 UUAGUCCUUACAAUAGGUCdTdT 226 GACCUAUUGUAAGGACUAAdTdT AL-DP-2107 60206018 113 UAGUCCUUACAAUAGGUCCdTdT 227 GGACCUAUUGUAAGGACUAdTdT AL-DP-21086252 6250 114 AUAUUCUAUAGCUGGACGUdTdT 228 ACGUCCAGCUAUAGAAUAUdTdTAL-DP-2109 6253 6251 115 UAUUCUAUAGCUGGACGUAdTdT 229UACGUCCAGCUAUAGAAUAdTdT AL-DP-2110 6254 6252 116 AUUCUAUAGCUGGACGUAAdTdT230 UUACGUCCAGCUAUAGAAUdTdT AL-DP-2111

TABLE 1a RSV L gene siRNA activity % inh % inh % inh % inh % inh RSV LRSV A2 RSV A2 RSV A2 RSV A2 RSV B gene duplex (5 nM) 500 pM 50 pM 5 pM(5 nM) AL-DP-2038 11 AL-DP-2031 15 AL-DP-2045 15 AL-DP-2050 15AL-DP-2056 16 AL-DP-2049 24 AL-DP-2026 82 AL-DP-2033 84 AL-DP-2048 84AL-DP-2027 86 AL-DP-2030 86 AL-DP-2034 86 AL-DP-2058 86 AL-DP-2066 86AL-DP-2036 87 AL-DP-2039 87 AL-DP-2047 87 AL-DP-2051 87 AL-DP-2040 88AL-DP-2055 88 AL-DP-2061 88 AL-DP-2029 89 AL-DP-2035 89 AL-DP-2069 89AL-DP-2028 90 AL-DP-2032 90 AL-DP-2063 90 AL-DP-2037 91 AL-DP-2059 91AL-DP-2065 91 AL-DP-2024 92 AL-DP-2053 92 84 79 76 74 AL-DP-2060 92AL-DP-2067 92 AL-DP-2068 93 AL-DP-2046 94 94 91 91 93 AL-DP-2057 94 9186 79 69 AL-DP-2064 94 86 76 67 83 AL-DP-2062 95 79 78 72 94 AL-DP-204196 76 73 69 94 AL-DP-2042 96 98 97 97 90 AL-DP-2052 96 84 76 69 87AL-DP-2043 97 86 79 75 94 AL-DP-2044 97 79 72 67 84 AL-DP-2054 97 79 7869 96

TABLE 1b RSV P gene siRNA Actual SEQ ID SEQ RSV P gene start Start_PosNO. Sense ID NO. Antisense duplex ID # 55 53 231 AAAUUCCUAGAAUCAAUAAdTdT250 UUAUUGAUUCUAGGAAUUUdTdT AL-DP-2000 56 54 232 AAUUCCUAGAAUCAAUAAAdTdT251 UUUAUUGAUUCUAGGAAUUdTdT AL-DP-2001 58 56 233 UUCCUAGAAUCAAUAAAGGdTdT252 CCUUUAUUGAUUCUAGGAAdTdT AL-DP-2002 59 57 234 UCCUAGAAUCAAUAAAGGGdTdT253 CCCUUUAUUGAUUCUAGGAdTdT AL-DP-2003 61 59 235 CUAGAAUCAAUAAAGGGCAdTdT254 UGCCCUUUAUUGAUUCUAGdTdT AL-DP-2004 322 320 236ACAUUUGAUAACAAUGAAGdTdT 255 CUUCAUUGUUAUCAAAUGUdTdT AL-DP-2005 323 321237 CAUUUGAUAACAAUGAAGAdTdT 256 UCUUCAUUGUUAUCAAAUGdTdT AL-DP-2006 324322 238 AUUUGAUAACAAUGAAGAAdTdT 257 UUCUUCAUUGUUAUCAAAUdTdT AL-DP-2007325 323 239 UUUGAUAACAAUGAAGAAGdTdT 258 CUUCUUCAUUGUUAUCAAAdTdTAL-DP-2008 426 424 240 AAGUGAAAUACUAGGAAUGdTdT 259CAUUCCUAGUAUUUCACUUdTdT AL-DP-2009 427 425 241 AGUGAAAUACUAGGAAUGCdTdT260 GCAUUCCUAGUAUUUCACUdTdT AL-DP-2010 428 426 242GUGAAAUACUAGGAAUGCUdTdT 261 AGCAUUCCUAGUAUUUCACdTdT AL-DP-2011 429 427243 UGAAAUACUAGGAAUGCUUdTdT 262 AAGCAUUCCUAGUAUUUCAdTdT AL-DP-2012 430428 244 GAAAUACUAGGAAUGCUUCdTdT 263 GAAGCAUUCCUAGUAUUUCdTdT AL-DP-2013431 429 245 AAAUACUAGGAAUGCUUCAdTdT 264 UGAAGCAUUCCUAGUAUUUdTdTAL-DP-2014 550 548 246 GAAGCAUUAAUGACCAAUGdTdT 265CAUUGGUCAUUAAUGCUUCdTdT AL-DP-2015 551 549 247 AAGCAUUAAUGACCAAUGAdTdT266 UCAUUGGUCAUUAAUGCUUdTdT AL-DP-2016 248 CGAUAAUAUAACAGCAAGAdTsdT 267UCUUGCUGUUAUAUUAUCGdTsdT AL-DP-1729 249 CGAUUAUAUUACAGGAUGAdTsdT 268UCAUCCUGUAAUAUAAUCGdTsdT AL-DP-1730

TABLE 1b RSV P gene activity % % % % % inhibition inhibition inhibitioninhibition RSV P gene inhibition RSV A2 RSV A2 RSV A2 RSV B duplex ID #(5 nM) 500 pM 50 pM 5 pM (5 nM) AL-DP-2000 3 AL-DP-2001 4 AL-DP-2002 7AL-DP-2003 98 93 92 84 97 AL-DP-2004 3 AL-DP-2005 7 AL-DP-2006 5AL-DP-2007 4 AL-DP-2008 7 AL-DP-2009 2 AL-DP-2010 7 AL-DP-2011 4AL-DP-2012 96 77 68 66 92 AL-DP-2013 98 85 76 75 89 AL-DP-2014 98 85 8168 66 AL-DP-2015 7 AL-DP-2016 98 88 82 75 94 AL-DP-1729 90 AL-DP-1730

TABLE 1c RSV N gene siRNA SEQ SEQ Actual ID ID RSV N gene start NO.Sense NO. Antisense DUPLEX ID # 3 1 GGCUCUUAGCAAAGUCAAGdTdT 2CUUGACUUUGCUAAGAGCCdTdT AL-DP-2017 5 269 CUCUUAGCAAAGUCAAGUUdTdT 277AACUUGACUUUGCUAAGAGdTdT AL-DP-2018 52 270 CUGUCAUCCAGCAAAUACAdTdT 278UGUAUUUGCUGGAUGACAGdTdT AL-DP-2019 53 271 UGUCAUCCAGCAAAUACACdTdT 279GUGUAUUUGCUGGAUGACAdTdT AL-DP-2020 191 272 UAAUAGGUAUGUUAUAUGCdTdT 280GCAUAUAACAUACCUAUUAdTdT AL-DP-2021 379 273 AUUGAGAUAGAAUCUAGAAdTdT 281UUCUAGAUUCUAUCUCAAUdTdT AL-DP-2022 897 274 AUUCUACCAUAUAUUGAACdTdT 282GUUCAAUAUAUGGUAGAAUdTdT AL-DP-2023 898 275 UUCUACCAUAUAUUGAACAdTdT 283UGUUCAAUAUAUGGUAGAAdTdT AL-DP-2024 899 276 UCUACCAUAUAUUGAACAAdTdT 284UUGUUCAAUAUAUGGUAGAdTdT AL-DP-2025

TABLE 1c RSV N gene activity % % % % % inhibition inhibition inhibitioninhibition RSV N gene inhibition RSV A2 RSV A2 RSV A2 RSV B Duplex ID #(5 nM) 500 pM 50 pM 5 pM (5 nM) AL-DP-2017 98 86 84 80 93 AL-DP-2018 2AL-DP-2019 5 AL-DP-2020 2 AL-DP-2021 3 AL-DP-2022 98 78 77 75 94AL-DP-2023 1 AL-DP-2024 7 AL-DP-2025 96 89 84 77 96

Example 2 In Vitro Assay and Virus Infection

Vero E6 cells were cultured to 80% confluency in DMEM containing 10%heat-inactivated FBS. For siRNA introduction, 4 μl of Transit-TKO wasadded to 50 μl of serum-free DMEM and incubated at room temperature for10 minutes. Then, an indicated concentration of siRNA was added tomedia/TKO reagent respectively and incubated at room temperature for 10minutes. This mixture was added to 200 μl of DMEM containing 10% FBS andthen to a monolayer of cultured cells. The cells were incubated at 37°C., 5% CO₂ for 6 hours. The RNA mixture was removed by gentle washingwith 1× Hank's Balanced Salt Solutions (HBSS) and 300 plaque-formingunits (pfu) per well of RSV/A2 (MOI=30) was added to wells and adsorbedfor 1 hour at 37° C., 5% CO₂. Virus was removed and the cells werewashed with 1×HBSS. Cells were overlaid with 1% methylcellulose in DMEMcontaining 10% FBS media, and incubated for 6 days at 37° C., 5% CO₂.Cells were immunostained for plaques using anti-F protein monoclonalantibody 131-2A.

Example 3 siRNA Delivery and Virus Infection In Vivo

Pathogen-free 4 week old female BALB/c mice were purchased from Harlan.Mice were under anesthesia during infection and intranasal instillation(i.n.). Mice were immunized by intranasal instillation with indicatedamount of siRNA, either uncomplexed, or complexed with 5 μl Transit TKO.150 μg of Synagis (monoclonal antibody clone 143-6C, anti-RSV F protein)and Mouse Isotype control (IgG1) were administered intraperitoneal(i.p.) four hours prior to RSV challenge (10⁶ PFU of RSV/A2). Ten miceper group were used. Animal weights were monitored at days 0, 2, 4, and6 post-infection. Lungs were harvested at day 6 post-infection, andassayed for RSV by immunostaining plaque assay.

Example 4 Immunostaining Plaque Assay

24-well plates of Vero E6 cells were cultured to 90% confluency in DMEMcontaining 10% heat inactivated FBS. Mice lungs were homogenized withhand-held homogenizer in 1 ml sterile Dulbecco's PBS (D-PBS) and 10 folddiluted in serum-free DMEM. Virus containing lung lysate dilutions wereplated onto 24 well plates in triplicate and adsorbed for 1 hour at 37°C., 5% CO₂. Wells were overlaid with 1% methylcellulose in DMEMcontaining 10% FBS. Then, plates were incubated for 6 days at 37° C., 5%CO₂. After 6 days, overlaid media was removed and cells were fixed inacetone:methanol (60:40) for 15 minutes. Cells were blocked with 5% dryMilk/PBS for 1 hour at 37° C. 1:500 dilution of anti-RSV F proteinantibody (131-2A) was added to wells and incubated for 2 hours at 37° C.Cells were washed twice in PBS/0.5% Tween 20. 1:500 dilution of goatanti-mouse IgG-Alkaline Phosphatase was added to wells and incubated for1 hour at 37° C. Cells were washed twice in PBS/0.5% Tween 20. Reactionwas developed using Vector's Alkaline Phosphatase substrate kit II(Vector Black), and counterstained with Hematoxylin. Plaques werevisualized and counted using an Olympus Inverted microscope.

Example 5 Treatment Assay

Mice were challenged with RSV (10⁶ PFU of RSV/A2) by intranasalinstillation at day 0 and treated with 50 μg of indicated siRNA,delivered by intranasal instillation, at the indicated times (day 1-4post viral challenge). 3-5 mice per group were used and viral titerswere measured from lung lysates at day 5 post viral challenge, aspreviously described.

Example 6 In Vitro Inhibition of RSV Using iRNA Agents

iRNA agents provided in Table 1 (a-c) were tested for anti-RSV activityin a plaque formation assay as described above. Results are shown inFIG. 1. Each column (bar) represents an iRNA agent provided in Table 1(a-c), e.g., column 1 is the first agent in Table 1a, second column isthe second agent and so on. Active iRNA agents were identified by the %of virus remaining Several agents were identified that showed as much as90% inhibition. The results are summarized in Table 1 (a-c).

In vitro dose response inhibition of RSV using iRNA agents wasdetermined. Examples of active agents from Table 1 were tested foranti-RSV activity in a plaque formation assay as described above at fourconcentrations. A dose-dependent response was found with active iRNAagents tested as illustrated in FIG. 2) and summarized in Tables 1(a-c).

In vitro inhibition of RSV B subtype using iRNA agents was tested asdescribed above. iRNA agents provided in FIG. 2 were tested at 5 nM foranti-RSV activity against subtype B as shown in FIG. 3. RSV subtype Bwas inhibited by the iRNA agents tested to varying degrees. Theseresults also are summarized in Table 1(a-c).

Example 7 In Vivo Inhibition of RSV Using iRNA Agents

In vivo inhibition of RSV using AL1729 and AL1730 was tested asdescribed above. Agents as described in FIG. 4 were tested for anti-RSVactivity in a mouse model. The iRNA agents were effective at reducingviral titers in vivo and more effective than a control antibody (Mab143-6c, a mouse IgG1 Ab that is approved for RSV treatment).

AL1730 was tested for dose-dependent activity using the methods providedabove. The agent showed a dose-dependent response as illustrated in FIG.5.

iRNA agents showing in vitro activity were tested for anti-RSV activityin vivo as outlined above. Several agents showed a reduction in viraltiters of >4 logs when given prophylactically as illustrated in FIG. 6.

iRNA agents showing in vitro and/or in vivo activity were tested foranti-RSV activity in vivo as in the treatment protocol outlined above.Several agents showed a reduction in viral titers of 2-3 logs as shownin FIG. 7 when given 1-2 days following viral infection.

Example 8 Sequence Analysis of Isolates Across Target Sequence

Growth of isolates and RNA isolation: Clinical isolates from RSVinfected patients were obtained from Larry Anderson at the CDC inAtlanta Ga. (4 strains) and John DeVincenzo at the University of Term.,Memphis (15 strains). When these were grown in HEp-2, human epithelialcells (ATCC, Cat# CCL-23) cells, it was noted that the 4 isolates fromGeorgia were slower growing than the 15 strains from Tennessee; hence,these were processed and analyzed separately. The procedure is brieflydescribed as follows:

Vero E6, monkey kidney epithelial cells (ATCC, Cat# CRL-1586) were grownto 95% confluency and infected with a 1/10 dilution of primary isolates.The virus was absorbed for 1 hour at 37° C., then cells weresupplemented with D-MEM and incubated at 37° C. On a daily basis, cellswere monitored for cytopathic effect (CPE) by light microscopy. At 90%CPE, the cells were harvested by scraping and pelleted by centrifugationat 3000 rpm for 10 minutes. RNA preparations were performed by standardprocedures according to manufacturer's protocol.

Amplification of RSV N gene: Amplification of the RSV N gene fragmentcontaining the ALN-RSV01 recognition site was performed using two stepRT-PCR.

First, RNA was reverse transcribed using random hexamers and SuperscriptIII Reverse transcriptase (Invitrogen, Carlsbad, Calif.) at 42° C. for 1hour, to generate a cDNA library. Next a 1200 nt gene specific fragmentwas amplified using the forward primer RSV NF:5′-AGAAAACTTGATGAAAGACA-3′ (SEQ ID NO: 285); and the reverse primer RSVNR: 5′-ACCATAGGCATTCATAAA-3′ (SEQ ID NO: 286) for 35 cycles at 55° C.for 30 sec followed by 68° C. for 1 min, using Platinum Taq polymerase(Invitrogen, Carlsbad, Calif.). PCR products were analyzed by 1% agarosegel electrophoresis.

Results: Sequence analysis of the first 15 isolates confirmed that thetarget site for ALN-RSV01 was completely conserved across every strain.Sequence alignments are provided in FIG. 8. Importantly, thisconservation was maintained across the diverse populations, whichincluded isolates from both RSV A and B subtypes. Interestingly, whenthe 4 slower-growing isolates were analyzed, we observed that one of the4 (LAP6824) had a single base mutation in the ALN-RSV01 recognition siteas shown in the top part of FIG. 9. This mutation changed the codingsequence at position 13 of the RSV N gene in this isolate from an A to aG (FIG. 9, bottom).

Conclusions: From 19 patient isolates, the sequence of the RSV N gene atthe target site for ALN-RSV01 has been determined. In 18 of 19 cases(95%), the recognition element for ALN-RSV01 was determined to be 100%conserved. In one of the isolates, there was detected a single basealteration changing the nucleotide at position 13 from an A to a Gwithin the RSV N gene. This alteration creates a single G:U wobblebetween the antisense strand of ALN-RSV01 and the target sequence asshown in FIG. 9, bottom. Based on an understanding of the hybridizationpotential of such a G:U wobble, ALN-RSV01 is predicted to be effectivein silencing the RSV N gene in this isolate.

Example 9 Synthesis and Purification of ALN-RSV01

As shown in FIG. 10, the process for manufacturing the ALN-RSV01 drugsubstance consists of synthesizing the two single strandoligonucleotides (sense and antisense) by conventional solid phasesynthesis using 3′-O-(2-cyanoethyl)phosphoramidite chemistry with the5′-hydroxyls protected with 4,4′-dimethoxytriphenylmethyl(dimethoxytrityl, DMT) groups and tert-butyldimethylsilyl (TBDMS)protection on the 2′-hydroxyls of the ribose nucleosides. The crudesingle strand oligonucleotides were cleaved from the solid support,de-protected in a two-step process and purified by preparative anionexchange high performance liquid chromatography (AX-HPLC). The twosingle strands were combined in an equimolar ratio followed by annealingand lyophilization to produce the ALN-RSV01 drug substance.

Solid Phase Synthesis: Assembly of an oligonucleotide chain by thephosphoramidite method on a solid support, such as controlled pore glass(CPG) or polystyrene followed the iterative process outlined in FIG. 11.The synthesis of ALN-RSV01 sense and antisense single strandintermediates was carried out on support loaded with 5′-dimethoxytritylthymidine. Each intermediate was assembled from the 3′ to the 5′terminus by the addition of protected nucleoside phosphoramidites and anactivator. All the reactions took place on the derivatized support in apacked column. Each elongation cycle consisted of four distinct steps.

5′-Hydroxyl Deprotection (Detritylation, FIG. 11 step A): In thebeginning of the synthesis the DMT-thymidine support was subjected toremoval of the acid labile 4,4′-dimethoxytrityl protecting group fromthe 5′-hydroxyl. Each cycle of the synthesis thereafter commenced withremoval of the corresponding DMT protecting group from the 5′ oxygenatom of the support-bound oligonucleotide (FIG. 11 step A). This wasaccomplished by treatment with a solution of dichloroacetic acid intoluene. Following detritylation the support-bound material was washedwith acetonitrile in preparation for the next reaction.

Coupling (FIG. 11 step B): The elongation of the growing oligonucleotidechain was achieved by reaction of the 5′-hydroxyl group of thesupport-bound oligonucleotide with an excess of a solution of theprotected nucleoside phosphoramidite, in the presence of the activator5-ethylthio-1H-tetrazole. The amidite required in each step wasdetermined by the oligonucleotide sequence described in Table 1c. Thisresulted in the formation of a phosphite triester internucleotidelinkage. After allowing sufficient time for the coupling reaction tocomplete, excess phosphoramidite and activator was rinsed from thereactor using acetonitrile.

Oxidation (FIG. 11 step C): The newly created phosphite triester linkagewas then oxidized by treatment with a solution of iodine in pyridine inthe presence of water. This resulted in the formation of thecorresponding phosphotriester bond (FIG. 11 step C). After the oxidationwas complete, the excess reagent (iodine in pyridine/water) was removedfrom the column by rinsing the support with acetonitrile.

Capping (FIG. 11 step D): Although the coupling reaction proceeds invery high yield it is not quite quantitative. A small proportion(typically less than 1%) of the 5′-hydroxy groups, available in anygiven cycle, fails to couple with the activated phosphoramidite. Inorder to prevent reaction during subsequent cycles these sites wereblocked by using capping reagents (acetic anhydride and Nmethylimidazole/2,6 lutidine/acetonitrile). As a result 5′-O-acetylated(capped') support-bound oligonucleotide sequences were formed.

Cleavage and De-protection: Reiteration of this basic four-step cycleusing the appropriate protected nucleoside phosphoramidites allowedassembly of the entire protected sequence. The DMT group protecting thehydroxyl at the 5′-terminus of the oligonucleotide chain was removed.The crude oligonucleotide was cleaved from the solid support by aqueousmethylamine treatment with concomitant removal of the cyanoethylphosphate protecting group. The support was removed by filtration andwashed with dimethyl sulfoxide. The cleavage solution and wash werecombined and held at room temperature or elevated temperatures tocomplete the deprotection of the exocyclic amino groups (benzoyl,isobutyryl, and acetyl) as shown in FIG. 12 step A. The 2′-O-TBDMSprotecting groups were then cleaved using a solution ofpyridine-hydrogen fluoride to yield the crude oligonucleotide (FIG. 12step B). At the completion of the deprotection the solution was dilutedwith aqueous buffer and subjected to the purification step.

AX-HPLC Purification: Purification of each crude product solution wasaccomplished by AX-HPLC. A solution of crude product was loaded onto thepurification column packed with Source 15Q media. The purification runwas performed using sodium phosphate buffered eluents containingapproximately 10% acetonitrile. A sodium chloride gradient was used toelute the oligonucleotide from the column. The purification was carriedout at elevated temperatures (65-75° C.). The elution profile wasmonitored by UV absorption spectroscopy. Fractions were collected andpooled. Pools containing product at target purity levels were subjectedto the next step in the process. Fractions that did not meet theacceptance criteria were, in some instances, repurified.

Desalting: The oligonucleotide solutions were concentrated usingtangential flow filtration (TFF) using a polyethersulfone (PES) membranecassette with a nominal 1,000 molecular weight cut-off. The retentatefrom the concentration step was pH adjusted and diafiltered with waterto remove salts and solvents used in the AX-HPLC purification. Thedesalted product solution (retentate) was sometimes further concentratedby TFF before transfer to the next step.

Duplex Formation: The ultrafiltered solutions of the sense and antisensestrand were combined in the desired proportions to form an equimolarmixture of the two intermediates. The required amounts of each singlestrand oligonucleotide were calculated based on UV assay and theirmolecular weights. To assure better control, the calculated amount ofthe first strand was mixed with less than the calculated amount of thesecond strand. AX-HPLC analysis of a sample of that mixture showed awell-resolved peak for the excess of the first strand together with apeak for the duplex. An additional amount of the second strand was addedand a sample was analyzed again. This process was repeated until excessof one of the strands is was determined to be ≦1 area % as judged by theHPLC chromatogram. The solution was then heated and cooled undercontrolled conditions to anneal the duplex.

Freeze Drying: The duplex solution was filtered through a 0.2-micronfilter before loading into disposable single use trays for bulk drying.The filtered product solution was freeze dried using a cycle consistingof three steps: (1) a freeze step, (2) primary drying at 0° C., and (3)secondary drying at 25° C. The result of this process is a lyophilizedpowder, i.e., a powder produced by the process that includes the stepsof freezing a liquid and, drying the frozen liquid product under vacuumto remove by sublimation some or all of the frozen water.

Container Closure System: The lyophilized ALN-RSV01 drug substance waspackaged in clean high-density polyethylene bottles with screw closures,labeled and stored in a freezer at −10 to −25° C. until shipment. Insome instances, a moisture barrier bag was added to the packaging of theinventory. The selected bag (Model LF4835W from Laminated Films &Packaging) has three layers (white PET, foil, and polyethylene) and isspecifically recommended as a barrier for oxygen and moisture.

Drug Finishing: ALN-RSV01 drug substance was delivered to a sterilefill/finish site as a lyophilized powder in sealed containers. Eachcontainer held a known weight of ALN-RSV01 drug substance. The bulkweight, the duplex purity, and the water content value were used tocalculate the ALN-RSV01 drug substance available for formulation. As thedrug substance is hygroscopic, whole containers were allocated for themanufacturing process. The size of the containers used allowed drugallocation to be close to the target lot size. The phosphate buffersolution was prepared to the required composition in a quantity inexcess of that required to prepare the target lot size. The pH of thebuffer was adjusted to 7.4±0.7. Allocated ALN-RSV01 drug substance, inwhole vials, was dissolved into 80% of the target batch volume ofphosphate buffer solution. An in-process sample was taken and thepotency measured by UV/SEC. Using this assay value the theoretical batchsize was calculated to give 100% potency. Using the remaining preparedbuffer the lot was brought to this theoretical volume. The pH wasmonitored and adjusted as needed to 6.6+1.0. The lot was thenaseptically filtered through two 0.22 μm sterile filters in series,filled into individual, sterile vials, stoppered, sealed, inspected(100% visual), and labeled. All vials were stored at 2-8° C.

Formulation Development: ALN-RSV01 drug product was formulated to a pHof 6.6 with sodium phosphate buffer. Phosphoric acid and sodiumhydroxide were available for pH adjustment as needed. The formulationwas near isotonicity, therefore there was no need to use sodium chlorideto adjust osmolality. Osmolality of the ALN-RSV01 drug product used forintranasal administration or inhalation preferably ranges between200-400 mOsm/kg.

Each vial of ALN-RSV01 drug product contains a volume of 0.5 mL. Theproduct was filled into clear Type I glass vials sealed withTeflon-coated butyl rubber stoppers with aluminum flip-off overseals.All vials were maintained at 2-8° C. and were warmed to room temperatureprior to use. In some instances, dilutions of drug product were preparedin normal saline by pharmacy staff.

Description and Composition of the Drug Product: ALN-RSV01 drug productwas formulated as an aqueous solution in 50 mM phosphate buffer, pH 6.6at a nominal concentration of 150 mg/mL. The quantitative composition ofthe ALN-RSV01 drug product is shown in Table 3. The weight shown forformulation reflects pure, anhydrous oligonucleotide. The amount ofactive ingredient per batch was calculated to account for the “as is”purity as determined by UV, ALN-RSV01 area value by SEC and the moisturecontent.

TABLE 3 quantitative composition of the ALN-RSV01 drug product QuantityIngredient per mL Function ALN-RSV01   150 mg Active Ingredient DibasicSodium 11.42 mg Buffer Phosphate Heptahydrate Monobasic Sodium Phosphate 1.01 mg Buffer Monohydrate Phosphoric Acid q.s. pH Adjustment SodiumHydroxide q.s pH Adjustment Water for Injection q.s. to 1 mL Vehicle

Stability Studies: ALN-RSV01 (Lot# R01) was evaluated after initial,one, two, three, six and nine months of storage and found to bechemically stable using stability indicating methods such as denaturingAX-HPLC and SEC. Follow on studies confirmed stability of an aqueousbuffered solution of the drug substance when stored at 2° C.-8° C. Thelyophilized drug substance stored at −20° C. is expected to be at leastas stable as the aqueous buffered solution. As used herein, “stable”means resistant to chemical changes that preclude product use in humansubjects. Stability can be assessed by measuring stability and purityusing methods that include denaturing AX-HPLC and SEC to providemeasures of the overall proportion of the drug product comprised of thesense and antisense strands, as well as the fraction of the drug productthat is found in duplex form. Other measures of stability include one ormore of: tests for pyrogens, analysis of water content, tests of the Tm,i.e., the parameter that addresses the quality of the duplex, and assayvalues for inhibition of RSV gene expression, drop in viral titre, etc.using, e.g., tests exemplified in the working examples.

Compatibility with BD AccuSpray™ Nasal Spray System: A phase 2a clinicalstudy was conducted by nasal instillation using the commerciallyavailable Becton-Dickinson Accuspray™ nasal spray system. Compatibilityof ALN-RSV01 drug product was confirmed by evaluating the stability ofALN-RSV01 drug product in contact with the system over a fourteen-dayperiod, both in ambient and refrigerated (2-8° C.) conditions. Nodegradation was observed upon storage of up to 14 days at 10 and 150mg/mL, in ambient and refrigerated conditions as measured by appearance,SEC, stability indicating denaturing AX-HPLC, pH, osmolality and UVassay.

Example 9 Silencing Data on Isolates

Methods: Vero E6 cells were cultured to 80% confluency in DMEMcontaining 10% heat-inactivated FBS. For siRNA introduction, 4 μl ofTransit-TKO was added to 50 μl of serum-free DMEM and incubated at roomtemperature for 10 minutes. Then, indicated concentration of siRNA wasadded to media/TKO reagent respectively and incubated at roomtemperature for 10 minutes. RNA mixture was added to 200 μl of DMEMcontaining 10% FBS and then to cell monolayer. Cells were incubated at37° C., 5% CO₂ for 6 hours. RNA mixture was removed by gentle washingwith 1× Hank's Balanced Salt Solutions (HBSS) and 300 plaque-formingunits (pfu) per well of RSV/A2 (MOI=30) was added to wells and adsorbedfor 1 hour at 37° C., 5% CO₂. Virus was removed and cells were washedwith 1×HBSS. Cells were overlaid with 1% methylcellulose in DMEMcontaining 10% FBS media, and incubated for 6 days at 37° C., 5% CO₂.Cells were immunostained for plaques using anti-F protein monoclonalantibody 131-2A.

Results: Silencing by ALN-RSV01 was seen for all isolates as shown inTable 4.

TABLE 4 silencing of isolates by ALN-RSV01 Isolate ALN-RSV01 2153 name %plaques remaining % plaques remaining RSV/A2 4.49 80.34 RSV/96 5.3687.50 RSV/87 10.20 79.59 RSV/110 5.41 81.08 RSV/37 4.80 89.60 RSV/672.22 91.67 RSV/121 6.25 82.50 RSV/31 4.03 96.77 RSV/38 2.00 92.67 RSV/985.13 91.03 RSV/124 3.74 90.37 RSV/95 7.32 64.02 RSV/32 5.45 92.73 RSV/918.42 95.79 RSV/110 12.07 94.83 RSV/54 1.90 89.87 RSV/53 7.41 94.07RSV/33 7.69 95.19

Conclusion: All clinical isolates tested were specifically inhibited byALN-RSV01 by greater than 85%. No isolates were significantly inhibitedby the mismatch control siRNA 2153.

Example 10 Silencing in Plasmid Based Assay

Methods: A 24-well plate was seeded with HeLa S6 cells and grown to 80%confluence. For each well, 1 μg of RSV N-V5 plasmid was mixed with asiRNA (at indicated concentration), in 50 ul OPTI-MEM which then wasadded to a Lipofectamine 2000 (Invitrogen)-Optimem mixture preparedaccording to manufacturer's instructions. This mixture was incubated for20 minutes at room temperature to allow time for complex formationbetween the siRNA and the Lipofectamine-Optimem components. Thecomplexed mixture was added complex to cells and incubated at 37° C.overnight. The media was removed, cells were washed withphosphate-buffered saline (PBS) and then lysed by the incubation with 50ul Lysis buffer (RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mMEDTA, 0.5% Na deoxycholate, 1% NP-40, 0.05% SDS) for 1-2 min. Lysateswere analyzed and inhibition of RSV-N protein expression was quantifiedby measuring the level of RSV-N protein in cell lysates, as detected byWestern blotting with an anti-V5 antibody.

Results: Transient plasmid expression was shown to be an effective assayfor RNAi agents (Table 5).

TABLE 5 Silencing as measured in a plasmid based assay siRNAConcentration Protein % Activity % 1 ALN-RSV01 10 nM 0 100 2 ALN-RSV01 1nM 0 100 3 ALN-RSV01 100 pM 0 100 4 ALN-RSV01 10 pM 11.78 88.22 5ALN-RSV01 1 pM 70.63 29.37 6 ALN-RSV01 100 fM 72.7 27.3 7 Control PBS100 0 8 2153 10 nM 94.54 4.5

Conclusions: siRNA 2017 (ALN-RSV01) was shown to specifically anddose-dependently inhibit the production of RSV N protein whentransiently cotranfected with a plasmid expressing the RSV N gene.Inhibition is not observed using mismatch control siRNA 2153.

Example 11 Silencing of RSV Via Aerosol Delivery of siRNA

Method: A 2 mg/ml solution of AL-DP-1729 or AL-DP-1730 was delivered vianebulization using an aerosol device for a total of 60 sec. Viralsamples were prepared from lung as described above and measured using anELISA instead of a plaque assay. The ELISA measures the concentration ofthe RSV N protein in virus-infected cells obtained from mouse lunglysates.

Methods: Lung lysate was diluted 1:1 with carbonate-bicarbonate buffer(NaHCO₃ pH 9.6) to a working concentration of 6-10 μg/100 μL, added toeach test well and incubated at 37° C. for 1 hour or overnight at 4° C.Wells were washed 3× with PBS/0.5% Tween 20 then blocked with 5% drymilk/PBS for 1 hour at 37° C. or overnight at 4° C. Primary antibody (Fprotein positive control=clone 131-2A; G protein positivecontrol=130-2G; negative control=normal IgG1κ, (BD Pharmingen, cat.#553454, test sera, or hybridoma supernatant) was added to the wells ata final dilution of 1:1000, and incubated at 37° C. for 1 hour orovernight at 4° C. Wells were washed 3× with PBS/0.5% Tween 20.Secondary antibody (Goat Anti-mouse IgG (H+ L) whole molecule-alkalinephosphatase conjugated) was added to the wells at a final dilution of1:1000 (100 μl/well) and incubated at 37° C. for 1 hour or overnight at4° C. The wells were washed 3× with PBS/0.5% Tween 20, after which time200 μl of Npp (Sigmafast) substrate (Sigma Aldrich N2770) made accordingto manufacturer's instructions was added to the wells. This mixture wasincubated for 10-15 at 37° C., and absorbances at OD 405/495 weremeasured.

Conclusion: Delivery of RSV specific siRNA decreases the levels of RSV Nprotein in mouse lungs as compared to the mismatch control siRNA (FIG.13 a-b).

Example 12 In Vivo Inhibition at Day −3-Prophylaxis

Method: In vivo prophylaxis was tested using the in vivo methoddescribed above except that the siRNA is delivered at different timesprior to infection with RSV from 3 days before to 4 hrs before. Resultswere obtained for AL-DP-1729 (active) and AL-DP-1730 (mismatch control).

Results: Active siRNA delivered intranasally up to 3 days prior to viralchallenge show specific and significant silencing in vivo as shown inFIG. 14.

Example 13 Nebulization of ALN-RSV01 with Pari eFlow® Device

Droplet Size and Analytical Integrity

Methods: A 150 mg/ml solution of ALN-RSV01 (in 2 mls of PBS) was filledinto the PARI EFLOW® electronic device and run until nebulization wascompleted and all aerosol was collected and allowed to condense in apolypropylene tube. Aliquots of material post nebulization were analyzedto determine geometric droplet size distribution by laser diffraction(Malvern MasterSizerX) under standard conditions. Aliquots of materialpre and post nebulization were analyzed to determine analyticalintegrity by a stability using anion exchange HPLC methodology.

Results: Aerosolized ALN-RSV01 had a Mass Median Diameter (MMD) of 3.1μm, a Geometric Standard Deviation (GSD) of 1.6 and a total respirablefraction of 85% (i.e., % particles <5 μm) confirming that a 75 mg/mlsolution could be aerosolized to yield respirable material withappropriate particle size. Comparison to control samples of ALN-RSV01formulation which were not nebulized showed matching chromatograms,demonstrating that the oligonucleotide can be nebulized by EFLOW®without degradation.

Biological Activity: A 25 mg/ml solution of ALN-RSV01 (in 1 ml of PBS)was prepared, 100 μl was removed (pre-nebulization aliquot) prior tonebulization with the PARI EFLOW® electronic device, and 500 ul of thenebulized solution was collected after condensing by passage over an icebath into a chilled 50 ml conical tube (post-nebulization aliquot).Serial dilutions of both aliquots were tested in our in vitrotransfection/infection plaque assay as previously described with theexception that siRNA was complexed with lipofetamine-2000.

Results: siRNA pre and post nebulization efficiently inhibited RSV viralreplication in a Vero cell plaque assay. The degree of inhibition wasalmost identical between the two samples and showed a dose responseleading to >80% silencing at the highest siRNA concentrations confirmingthat nebulized ALN-RSV01 maintains biological activity. Results areshown in FIG. 15.

Example 14 Inhalable siRNAs: ALN-RSV01

To investigate the in vivo effects of aerosolization and delivery byinhalation of siRNAs targeting RSV as well as the pharmacokineticproperties of inhaled siRNAs, a double-blind, randomized,placebo-controlled, evaluation study in human adult subjects wasperformed. The study measured routine bloods and clinical observations,inflammatory biomarkers, tolerability and plasma pharmacokinetics. Asused in this specification “inhalation” refers to administration of adosage form that is formulated and delivered for topical treatment ofthe pulmonary epithelium. As described above, an inhalable dosage formcomprise particles of respirable size, i.e., particles that aresufficiently small to pass through the mouth or nose and larynx uponinhalation and into the bronchi and alveoli of the lungs.

In the study, ascending doses of aerosolized ALN-RSV01 or placebo wereadministered once daily by inhalation for 3 consecutive days to 4cohorts of 12 subjects each with 8 subjects receiving ALN-RSV01 and 4subjects receiving placebo in each cohort for a total of 48 subjects.ALN-RSV01 maximum solubility concentration in the finished product is150 mg/mL. Therefore, a 150 mg/ml solution of ALN-RSV01 was diluted tothe appropriate concentration and filled into the PARI EFLOW® electronicdevice and run until nebulization was completed.

Blood samples evaluated for pharmacokinetics (PK) included pre dose andpost dose at 2, 5, 15, and 30 minutes, 1 hour and 24 hours on Day 0 andpost third dose at 2, 5, 15, and 30 minutes, 1 hour and 24 hours afterthe third dose (13 samples per subject). Urine collection for PKincluded: pre dose and post third dose at 0-6 hours, 6-12 hours and12-24 hours.

Plasma ALN-RSV01 concentrations, and derived parameters (C_(pre),C_(max), t_(max), t_(1/2), CL/F, V_(d)/F, AUC_(last)) were evaluated forPK.

ALN-RSV01 has previously been evaluated for toxicity by inhalationadministration in rats and monkeys at doses as high as 36 mg/kg/day and30 mg/kg/day, respectively. The highest dose to be administered in thesingle dose part of the current study was 210 mg/day (or 3 mg/kg,assuming 70 kg body weight). On a mg/kg basis, this dose isapproximately 10 fold lower than the doses given previously to rats andmonkeys.

The initial doses in this study were 7.0 mg, 21.0 mg and 70.0 mgproviding a safety margin of about 300 fold, 100-fold and 30 fold,respectively.

Dose levels for the multiple dose part of the study were 7.0 mg, 21.0mg, 70.0 mg and 210 mg, given as a daily delivered dose (DD) for threeconsecutive days.

The highest dose to be administered in the single dose part of thecurrent study was chosen at 210 mg/day (or 3 mg/kg, assuming 70 kg bodyweight).

Study drug exposure duration in the multiple dose part of the study waschosen to be 3 days, with once daily dosing, based on the intendedtherapeutic dosing duration which is likely to be short due to the acutenature of RSV infections.

Pulmonary Function Tests

PFT were conducted at screening to identify healthy volunteers withrespect to capacities and flow-rates. PFT provides an objective methodfor assessing the mechanical and functional properties of the lungs andchest wall. PFT measures:

-   -   Lung capacities e.g., Slow Vital Capacity (SVC) and Force Vital        Capacity (FVC), which provide a measurement of the size of the        various compartments within the lung    -   Volume parameters (e.g., FEV1) and flow-rates (e.g., FEF25-75),        which measure maximal flow within the airways

Serial evaluations of pulmonary function after inhalation of ALN-RSV01or placebo were conducted. Additional PFT testing was conducted on Day 0at pre-dose (about −30 min) and at 30 min and 2 h, 6 h, and 12 h on Days1, 1 and 2 at the same time as pre-dose on Day 0.

PFT provides lung capacities and flow-rates. The SVC is the volume ofgas slowly inhaled when going from complete expiration to completeinhalation. The FVC is the volume expired when going from completeinhalation to complete exhalation as hard and fast as possible. The FEV1is the amount expired during the first second of the FVC maneuver. TheForced Expiratory Flow (FEF25-75) is the average expiratory flow overthe middle half of the FVC. SVC, FVC, FEV1 and FEF25-75 was measuredaccording the ATS/ERS guidelines. In this study, FEV1 was the mainparameter.

As shown in FIG. 16, no significant change in lung function was seen onaerosol administration of ALN-RSV01.

Plasma

For single dosing, blood samples were collected for the analysis ofALN-RSV01 in plasma at pre dose and post dose (post nebulization) at 2,5, 15, and 30 minutes, 1 hour and 24 hours on Day 0 (7 samples pervolunteer).

For multi-dosing, blood samples were collected for analysis of ALN-RSV01in plasma at pre-dose and at 2, 5, 15 and 30 min, 1 h, and 24 h postfirst-dose on Day 0 (post nebulization), and at 2, 5, 15, 30 min, 1 h,and 24 h after the third dose (post dose nebulization of third dose).

Blood samples of 5 mL each were taken via an indwelling intravenouscatheter or by direct venipuncture into tubes containing K3EDTA as theanticoagulant. In case of sampling through the intravenous catheter, thefirst 1 mL of blood was discarded in order to prevent any dilution ofblood with heparin used to flush the catheter.

Results

A safe and well tolerated regimen of ALN-RSV01 has been defined forfurther clinical development. To this end the data show that plasmaexposure for a given dose in man is greater than in nonhuman primates.See FIG. 17. While single dose administration at 3 mg/kg equivalent wasassociated with a greater incidence of a flu-like adverse event (cough,headache, non-cardiac chest pain, pharyngo-laryngeal pain and chills)relative to placebo multi-dose administration of AL-DP 2017 was safe andwell-tolerated when given once daily for 3 days up to 0.6 mg/kg perdose. There was also no evidence of neutrophil leucocytosis after multidosing of AL-DP-2107 in the highest dose cohort (0.6 mg/kg). See FIG.18.

Example 15 A Split Dose of ALN-RSV01 Reduced RSV Titer Levels In Vivo

A fixed dose (120 μg) of ALN-RSV01 was administered to rodentsintranasally 4 hours prior to RSV instillation (10⁶ pfu at timepointzero). Mice were then administered 120 μg of ALN-RSV01 intranasally onthe first, second or third day following instillation, or in threeadministrations split equally over days 1, 2, and 3 followinginstillation. The dose administered over the course of three daysmaintained the same reduced RSV titer levels in the lung as observed bythe single dose of the siRNA on the first day following infection. SeeFIG. 19.

Example 16 RNAi-Specific Activity of RSV-Targeted siRNAs

Materials and Methods

Animals. Six- to eight-week old, pathogen-free female BALB/c mice werepurchased from Harlan Sprague-Dawley Laboratories (Indianapolis, Ind.).The mice were housed in microisolator cages and fed sterilized water andfood ad libitum.

Virus preparation, cell lines and viral titering. Vero E6 cells weremaintained in tissue culture medium (TCM) consisting of Dulbecco'sModified Eagle Medium (D-MEM, GIBCO Invitrogen, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah). RSV/A2and RSV/B1 were prepared in Vero E6 cells. Briefly, confluent Vero E6cells (American Type Culture Collection, Manassas, Va.) in serum-freeD-MEM, were infected with RSV at a multiplicity of infection (MOI) of0.1. The virus was adsorbed for 1 h at 37° C. after which TCM was added.Infected cells were incubated for 72-96 h at 37° C. until >90%cytopathic effect (CPE) was observed by light microscopy. Infected cellswere harvested by removal of the medium and replacement with a minimalvolume of serum-free D-MEM followed by three freeze-thaw cycles at −70and 4° C., respectively. The contents were collected and centrifuged at4000×g for 20 min at 4° C. to remove cell debris, and the titer wasdetermined by immunostaining plaque assay as previously described.Briefly, Vero E6 cells are infected with serial dilutions of stock RSV,adsorbed for 1 h at 37° C., then overlayed with 2% methylcellulose media(DMEM, supplemented with 2% fetal bovine serum, 1%antibiotic/antimycotic solution, 2% methylcellulose). After 5 days at37° C./5% CO₂, plates are removed and cells are fixed with ice-coldAcetone:Methanol (60:40). Cells are blocked with Powerblock, universalblocking reagent (Biogenix, San Ramon, Calif.), incubated with Anti-RSVF protein monoclonal antibody 131-2A dilute 1:200 (Millipore-Chemicon,),followed by Goat anti-mouse IgG whole molecule alkaline phosphatasesecondary antibody. The reaction was developed with Alkaline phosphatasesubstrate kit (Vector Black, Vector Laboratories, Burlingame, Calif.)and plaques were visualized and counted using a light microscope. ForRSV primary isolate cultures, samples were obtained from Dr. JohnDeVincenzo from the University of Tennessee, Memphis, Tenn. RSV isolateswere obtained from RSV infected children diagnosed by either aconventional direct fluorescent antibody (DFA) method or by a rapidantigen detection method in the Le Bonheur Children's Medical CenterVirology Laboratory in Memphis, Tenn. Nasal secretions were collected byaspiration, grown and passaged in HEp-2 cells and harvested at 90%cytopathic effect. Individual aliquots of supernatant containing RSVwere then subjected to nucleic acid extraction using QiAmp Viral RNAmini kit, according to the manufacturer's protocol (Qiagen, Valencia,Calif.). RSV isolates were also obtained from Mark Van Ranst from theUniversity of Leuven, Leuven, Belgium and Larry Anderson from theCenters for Disease Control and Prevention, Atlanta, Ga.

RSV-specific siRNA selection. Using one of the National Center forBiotechnology Information (NCBI) databases a Basic Local AlignmentSearch Tool (BLAST), was performed. In this analysis, a sequencecomparison algorithm is used to search sequence databases for optimallocal alignments to a query (2). In this case, the query is the 19 ntsequence comprising the sense or antisense strand of ALN-RSV01,excluding the dTdT overhang. The database, Reference Sequence (RefSeq),provides a comprehensive, integrated, non-redundant set of sequences,including genomic DNA, transcript (RNA), and protein products, and isupdated weekly (49). Only siRNAs that showed no significant homology toany sequence from the RefSeq database were selected for synthesis andfurther study.

In vitro RSV inhibition assay. Vero cells, in 24-well plates, were grownin a 5% CO₂ humidified incubator at 37° C. in DMEM supplemented with 10%fetal bovine serum (Life Technologies-Invitrogen, Carlsbad, Calif.), 100units/ml penicillin, and 100 g/ml streptomycin (BioChrom, Cambridge, UK)to 80% confluence. siRNAs were diluted to the indicated concentrationsin 50 μl Opti-MEM Reduced Serum Medium (Invitrogen). Separately, 3 μlLipofectamine 2000 (Invitrogen) was diluted in 50 μl Opti-MEM mixed andincubated for 5 minutes at room temperature. siRNA and lipofectaminemixtures were combined, incubated for 20-25 minutes at room temperature,then added to cells and incubated at 37° C. overnight. The mixture wasthen removed from cells and 200-400 plaque forming units of RSV/A2 wasincubated with cells for 1 hour at 37° C. The infected cells werecovered with methylcellulose media and incubated for 5 days at 37° C.and plaques visualized by immunostaining plaque assay as described.

In vivo screening of RSV-specific siRNAs. For the prophylaxis model,BALB/c mice were anesthetized by intraperitoneal (i.p.) administrationof 2,2,2-tribromoethanol (Avertin) and instilled intranasally (i.n.)with siRNA in a total volume of 50 μl of PBS. At 4 hours post siRNAinstillation, the mice were anesthetized and infected i.n. with 1×10⁶PFU of RSV/A2 in 50 μl. Prior to removal of lungs at day 4post-infection, anesthetized mice were exsanguinated by severing theright caudal artery. Lung tissue was collected in 1 ml ice coldphosphate-buffered saline (PBS; GIBCO Invitrogen). RSV titers from lungswere measured by immunostaining plaque assay. Lungs were homogenizedwith a hand-held Tissumiser homogenizer (Fisher Scientific, Pittsburg,Pa.) and lung homogenates were placed on ice for 5-10 minutes to allowdebris to settle. Clarified lung lysates were serially diluted 10-foldin serum-free D-MEM, added to 95% confluent Vero E6 cells cultured inD-MEM in 24-well plates (BD Falcon, San Jose, Calif.), and plaque assayswere performed as described above. For the treatment model, BALB/c micewere anesthetized as above and instilled i.n. with 1×10⁶ PFU of RSV/A2in 50 μl. At one, two, three or four days post viral infection, micewere reanesthetized and instilled i.n. with siRNA in 50 μl and thenviral concentrations were measured in the lungs on day 5 post infection,as described above.

siRNA generation. RNA oligonucleotides were synthesized usingcommercially available5′-O-(4,4′-dimethoxytrityl)′3′O-(2-cyanoethyl-N,N-diisopropyl)phosphoramiditemonomers of uridine (U), 4-N-benzyoylcytidine (C^(Bz)),6-N-benzoyladenosine (A^(bz)) and 2-N-isobutyrlguanosine (G^(iBu)) with2′-O-t-butyldimethylsilyl protected phosphoramidites according tostandard solid phase oligonucleotide synthesis protocols (13). Aftercleavage and de-protection, RNA oligonucleotides were purified byanion-exchange high-performance liquid chromatography and characterizedby ES mass spectrometry. To generate siRNAs from RNA single strands,equimolar amounts of complementary sense and antisense strands weremixed and annealed, and siRNAs were further characterized by CGE.

PBMC assay. To examine the ability of siRNAs to stimulate interferonalpha (IFNα) or tumor necrosis factor alpha (TNFα), human peripheralblood mononuclear cells (hPBMCs) were isolated from concentratedfractions of leukocytes (buffy coats) obtained from the Blood Bank Suhl,Institute for Transfusion Medicine, Germany. Buffy coats were diluted1:1 in PBS, added to a tube of Histopaque (Sigma, St. Louis, Mo.) andcentrifuged for 20 minutes at 2200 rpm to allow fractionation. Whiteblood cells were collected, washed in PBS, followed by centrifugation.Cells were resuspended in RPMI 1640 culture medium (Invitrogen)supplemented with 10% fetal calf serum, IL-3 (10 ng/ml) (Sigma) andphytohemagglutinin-P (PHA-P) (5 μg/ml) (Sigma) for IFNα assay, or withno additive for TNFα assay at a concentration of 1×10⁶ cells/ml, seededonto 96-well plates and incubated at 37° C., 5% CO₂. Controloligonucleotides siRNA AL-DP-5048 duplex: 5′-GUCAUCACACUGAAUACCAAU-3′(SEQ ID NO: 287) and 3′-CACAGUAGUGUGACUUAUGGUUA-5′ (SEQ ID NO: 288);siRNA AL-DP-7296 duplex: 5′-CUACACAAAUCAGCGAUUUCCAUGU-3′ (SEQ ID NO:289) and 3′-GAUGUGUUUAGUCGCUAAAGGUACA-5′ (SEQ ID NO: 290); siRNAAL-DP-1730 duplex: 5′-CGAUUAUAUUACAGGAUGAdTsdT-3′ (SEQ ID NO: 249) and3′-dTsdTGCUAAUAUAAUGUCCUACU-5′ (SEQ ID NO: 268); and siRNA AL-DP-2153duplex: 5′-GGCUCUAAGCUAACUGAAGdTdT-3′ (SEQ ID NO: 291) and3′-dTdTCCGAGAUUCGAUUGACUUC-5′ (SEQ ID NO: 292) Cells in culture werecombined with either 500 nM oligonucleotide, pre-diluted in OptiMEM(Invitrogen), or 133 nM oligonucleotide pre-diluted in OptiMEM andGeneporter, GP2 transfection reagent (Genlantis, San Diego, Calif.) forIFNα assay or N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammoniummethylsulfate (DOTAP) (Roche, Switzerland) for TNFα assay and incubatedat 37° C. for 24 hrs. IFNα and TNFα were measured using the BenderMedSystems (Vienna, Austria) instant ELISA kit according tomanufacturer's instruction.

In vitro and in vivo RACE. Total RNA was purified from either in vitrotransfected Vero E6 cells or from lungs harvested at day 5post-infection as described above, using Tryzol (Invitrogen), followedby DNase treatment and final processing using RNeasy, according tomanufacturer instructions (Qiagen). Five to ten microliters of RNApreparation from pooled samples was ligated to GeneRacer adaptor(5′-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3′ (SEQ ID NO: 293))without prior treatment. Ligated RNA was reverse transcribed using agene specific primer (cDNA primer: 5′-CTCAAAGCTCTACATCATTATC-3′ (SEQ IDNO: 294)). To detect RNAi specific cleavage products, two rounds ofconsecutive PCR were performed using primers complimentary to the RNAadaptor and RSV A2 N gene mRNA (GR 5′ primer:5′-CGACTGGAGCACGAGGACACTGA-3′ (SEQ ID NO: 295) and Rev Primer:5′-CCACTCCATTTGCTTTTACATGATATCC-3′ (SEQ ID NO: 296)) for the firstround, followed by a second round of nested PCR (GRN 5′ primer:GGACACTGACATGGACTGAAGGAGTA-3′ (SEQ ID NO: 297) and Rev N Primer:5′-GCTTTTACATGATATCCCGCATCTCTGAG-3′ (SEQ ID NO: 298)). Amplifiedproducts were resolved by agarose gel electrophoresis and visualized byethidium bromide staining. Specific cleavage products migrating at thecorrect size were excised, cloned into a sequencing vector and sequencedby standard method.

Sequence Analysis of Clinical Isolates for ALN-RSV01 Target SiteConservation. Amplification of the RSV N gene fragment containing theALN-RSV01 recognition site was performed using two-step RT-PCR. Briefly,RNA was reverse transcribed using random hexamers and Superscript IIIreverse transcriptase (Invitrogen) at 42° C. for 1 hr to generate a cDNAlibrary. A 1200 nucleotide gene specific fragment was amplified usingthe RSV N forward primer: 5′-AGAAAACTTGATGAAAGACA-3′ (SEQ ID NO: 285)and the RSV N reverse primer: 5′-ACCATAGGCATTCATAAA-3′ (SEQ ID NO: 286)for 35 cycles at 55° C. for 30 sec followed by 68° C. for 1 min usingPlatinum Taq polymerase (Invitrogen). PCR products were analyzed by 1%agarose gel electrophoresis. As a control, a laboratory strain of RSV Along was subjected to the identical procedures for analysis. PCRproducts were purified using QIAquick PCR purification kit (Qiagen)according to the manufacturer's protocol and sequenced using standardprotocols (Agencourt Bioscience, Beverly, Mass.). For each clone,forward and reverse sequence was obtained. Sequences were analyzed andaligned via Clustal W and ContigExpress using Vector NTI software(Invitrogen).

RSV viral genotyping. Genotyping of all 21 isolates received from MarkVan Ranst were performed as described previously (78). Genotyping of theremaining 78 (57 from Dr. John DeVincenzo, University of Tennessee, 13from Dr. Larry Anderson, Centers for Disease Control and Prevention, and8 from Dr. Jeffrey Kahn, Yale University) isolates was performed by Dr.Jeffrey Kahn's Laboratory, Department of Pediatrics, Yale University,New Haven, Conn. Analysis of the RSV G gene was performed by firstgenerating cDNA using random hexamers and M-MuLV reverse transcriptase(New England Biolabs, Beverly, Mass.) at 37° C. for 1 hr, followed byPCR amplification using G gene specific primers GTmF:5′-CCGCGGGTTCTGGCAATGATAATCTCAAC-3′ (SEQ ID NO: 299) and subgroupspecific G gene specific primers RSV A-GAR2:5′-GCCGCGTGTATAATTCATAAACCTTGGTAG-3′ (SEQ ID NO: 300) or RSV B-GBR:5′-GGGGCCCCGCGGCCGCGCATTAATAGCAAGAGTTAGGAAG-3′ (SEQ ID NO: 301) bydenaturing at 95° C. for 15 min, followed by 40 cycles of 95° C. for 1min, 60° C. for 1 min and 72° C. for 1 min, followed by a single 10 minextension at 72° C. using HotStar Taq DNA polymerase (Qiagen). PCRproducts were analyzed by 2% agarose gel electrophoresis. If anappropriate size PCR product was identified (RSV A: 1200 nt or RSV B:900 nt), the product was purified using QIAquick Extraction Kit (Qiagen)according to the manufacturer's protocol. Purified PCR products wereanalyzed by agarose gel electrophoresis and sequenced on a 3730 XL DNAAnalyzer (Applied Biosystems, Foster City, Calif.).

Nucleotide sequences were aligned manually and alignment confirmed usingClustal W. RSV A and RSV B isolates were distinguished by comparing Ggene nucleotide sequences and laboratory standards. Phylogeneticanalysis for RSV A isolates was performed using an aligned 417 ntsegment of the G gene corresponding to nucleotide position 5010 to 5426(GenBank Accession # M74568). Phylogenetic analysis of RSV B isolateswas performed using an aligned 288 nt segment of the G genecorresponding to nucleotide positions 5036 to 5323 (GenBank Accession#AF013254). Bootstrap datasets containing 1000 aligned permutednucleotide sequence sets were produced using SEQBOOT (PHYLIP 3.65).Maximum likelihood phylogenetic trees were obtained using DNAML (PHYLIP3.65) with default settings and the bootstrap datasets. CONSENSE (PHYLIP3.65) was used to produce an extended majority rule consensus tree andthe trees were MEGA4. Bootstrap values, isolate clustering and previousgenotype assignments were used to determine RSV genotypes (FIG. 26).

Results

Bioinformatic analysis of RSV genome and selection of ALN-RSV01. Thethree proteins contained within the nucleocapsid (nucleoprotein (N),phosphoprotein (P), and polymerase (L)) are required for various stepswithin the replication cycle of RSV (Collins, P., K. McIntosh, and R.Chanock. 1996. Respiratory Syncytial Virus, p. 1313-1351, Fields'Virology.), and are among the most highly conserved regions of the RSVgenome (Sullender, W. M. 2000. Respiratory syncytial virus genetic andantigenic diversity. Clin Microbiol Rev 13:1-15, table of contents;Sullender, W. M., L. Sun, and L. J. Anderson. 1993. Analysis ofrespiratory syncytial virus genetic variability with amplified cDNAs. JClin Microbiol 31:1224-31). To select appropriate siRNAs, GenBanksequences AF035006 (RSV/A2), AF013255 (RSV/B1), AY911262 (RSV/A Long),and D00736 (RSV/18537) were aligned using the Clustal W algorithm toidentify conserved 19mers amongst all RSV sequences analyzed. Todetermine uniqueness of each 19 mer across the human genome, a BasicLocal Alignment Search Tool (BLAST) analysis was performed against theReference Sequence (RefSeq) database. Only siRNAs with homology of 16nucleotides or fewer to any gene in the human genome were selected forfurther analysis.

Seventy siRNAs targeting the RSV N, P, and L genes were analyzed in aplaque inhibition assay and 19 exhibited >80% inhibition of plaqueformation versus a PBS control at siRNA concentrations of 20 nM (datanot shown). Of these 19 siRNAs, the siRNA designated “ALN-RSV01” (FIG.20) that targets the N gene, consistently demonstrated the highestanti-viral activity. Indeed, ALN-RSV01 showed an IC50 of 0.7 nM in theRSV plaque inhibition assay (FIG. 21).

ALN-RSV01 inhibition of RSV primary isolates. The relationship betweenclinical disease and molecular epidemiology of RSV is poorly understood,as several different genotypes cocirculate during most seasons, anddominating genotypes can vary from year to year. It is therefore crucialthat any prospective anti-viral agent targets the broadest possiblearray of identified genotypes. Based on the mechanism of RNAi, it ispredicted that sequence identity between an siRNA and its target,implies functional silencing. For this reason, a series of primaryisolates (genotype analysis, FIGS. 26A and 26B) taken from nasal washesof children with confirmed RSV disease, were sequenced across theALN-RSV01 recognition element. Of the RSV primary isolates sequenced,94% (89/95) showed absolute conservation across the ALN-RSV01 targetsite. The six isolates that were not 100% conserved each had a singlebase alteration within the ALN-RSV01 target site. Four had C-U mutationsat position 4 with respect to the 5′ end of the antisense strand ofRSV01, one had A-G mutation at position 7 and one had G-A mutation atposition 1. A subset of these 95 isolates was tested in the in vitroviral inhibition assay including one isolate with a mismatch at position4 and another with a mismatch at position 7 (Table 16). Of these, 12/12(100%) exhibited ˜70% inhibition at 80 nM ALN-RSV01 as compared to PBScontrol, and all had similar dose response curves for ALN-RSV01inhibition (FIG. 27).

TABLE 16 Name Target site SEQ ID NO: ALN-RSV01 GGCUCUUAGCAAAGUCAAG 302RSV A2 GGCUCUUAGCAAAGUCAAG 302 LAM 1238 GGCUCUUAGCAAAGUCAAG 302 LEO0713GGCUCUUAGCAAAGUCAAG 302 RUG0420 GGCUCUUAGCAAAGUCAAG 302 MOT0972GGCUCUUAGCAAAGUCAAG 302 BEN0819 GGCUCUUAGCAAAGUCAAG 302 JEN 1133GGCUCUUAGCAAAGUCAAG 302 HAN 1135 GGCUCUUAGCAAAGUUAAG 303 LAP 0824GGCUCUUAGCAAGGUCAAG 304 VA-37C GGCUCUUAGCAAAGUCAAG 302 VA-38CGGCUCUUAGCAAAGUCAAG 302 VA-54C GGCUCUUAGCAAAGUCAAG 302 RSV#32GGCUCUUAGCAAAGUCAAG 302

In vivo studies of ALN-RSV01. The BALB/c mouse is a well-establishedmodel for RSV infection, and was thus chosen as the in vivo system forevaluation of anti-viral efficacy of ALN-RSV01. Initially in aprophylaxis model, siRNA was administered intranasally (i.n.) to micefour hours prior to infection with 10⁶ pfu of RSV/A2. There was dosedependent inhibition of RSV/A2 replication in the lungs of mice, with a100 μg dose of ALN-RSV01 reducing titers between 2.5 to 3.0 log₁₀ pfu/glung as compared to either PBS controls or a non-specific siRNA (FIG.22A). Fifty and 25 μg doses yielded reductions of approximately 2.0 and1.25 log₁₀ pfu/g, respectively (FIG. 22A).

To evaluate the efficiency of viral inhibition in a treatment paradigm,ALN-RSV01 was delivered i.n., in single or multiple daily doses at 1, 2and/or 3 days post infection. When delivered as a single dose, the mostefficacious silencing by ALN-RSV01 occurred in prophylactic (−4 h)dosing in a dose-dependent fashion. As compared to the mismatch controlAL-DP-1730, administration of 120 μg of ALN-RSV01 as a singleprophylactic dose resulted in maximal viral inhibition, decreasing lungconcentrations down to background levels in this assay. When ALN-RSV01was administered in a treatment regime as a single dose following viralinoculation, anti-viral efficacy was maintained in a dose-dependentmanner but found to decrease as a function of time of dosing post viralinfection (FIG. 22B). Indeed, by Day 3 post infection, single doses ashigh as 120 μg did not yield any significant viral inhibition. However,when multiple 40 μg doses of ALN-RSV01 were delivered daily on days 1,2, and 3, the efficiency of silencing was maintained and viral titerswere again reduced to background levels (FIG. 22B).

To further explore alternative dosing paradigms that could be employedin future clinical studies, additional multi-dose regimens wereevaluated. To this end, RSV-infected mice were treated over a 12 hourperiod with ALN-RSV01 either in a 2× per day or 3× per day dose regimen.Interestingly, this multiple daily dose regimen of the RSV-specificsiRNA (40 μg 3×/day) was found to be as efficacious as a single 120 μgdose (FIG. 22C). In aggregate, these data show that a multi-dosetreatment regimen of ALN-RSV01 can provide maximal anti-viral efficacyin a fashion readily applicable to human clinical studies in relevantpatient populations.

ALN-RSV01 and cytokine induction. Many nucleic acids, includingdouble-stranded RNA (dsRNA), single-stranded RNAs (ssRNA) and siRNAshave been shown to stimulate the innate immune response through avariety of RNA binding receptors (Robbins, M., A. Judge, L. Liang, K.McClintock, E. Yaworski, and I. MacLachlan. 2007. 2′-O-methyl-modifiedRNAs act as TLR7 antagonists. Mol Ther 15:1663-9). This stimulation canbe monitored in vitro in a peripheral blood mononuclear cell (PBMC)assay (Sioud, M. 2005). Induction of inflammatory cytokines andinterferon responses by double-stranded and single-stranded siRNAs issequence-dependent and requires endosomal localization. (J Mol Biol348:1079-90.). While an immunostimulatory property of an siRNA could actsynergistically with an RNAi-mediated mechanism for the treatment of aviral infection, such a feature might also confound interpretation ofresults related to an siRNA treatment strategy. Accordingly, ALN-RSV01was evaluated for its ability to stimulate IFNα and TNFα in vitro byincubating with freshly purified peripheral blood mononuclear cells(PBMCs) as previously described (Hornung, V., M. Guenthner-Biller, C.Bourquin, A. Ablasser, M. Schlee, S. Uematsu, A. Noronha, M. Manoharan,S. Akira, A. de Fougerolles, S. Endres, and G. Hartmann. 2005.Sequence-specific potent induction of IFN-alpha by short interfering RNAin plasmacytoid dendritic cells through TLR7. Nat Med 11:263-70.). Highconcentrations of ALN-RSV01 were used in these assays (133 nM),exceeding the IC₅₀ for anti-viral effect by over 100-fold. After 24hours, only modest levels of both IFNα and TNFα were detected by ELISA,with an average of approximately 147 pg/ml of IFNα (FIG. 23A) and 1500pg/ml of TNFα (FIG. 23B) induced, as compared to media alone controls

To verify that the antiviral activity of ALN-RSV01 was not influenced bythis modest induction of cytokines, two non-RSV specific siRNAspreviously shown to more significantly induce either IFNα or TNFα, wereassayed in our in vivo BALB/c mouse model. Neither AL-DP-1730, a TNFαinducer (FIG. 24A) nor AL-DP-2153, a IFNα inducer (FIG. 24B) inhibitedRSV/A2 when administered intranasally (100 μg) into mice, as compared tothe strong inhibition observed when delivering ALN-RSV01 (FIG. 24C).Importantly, even 10 fold higher doses of AL-DP-1730 had no effect onRSV levels when delivered prophylactically, 4 hrs prior to infection(data not shown). These data support the conclusion that ALN-RSV01antiviral effects are mediated via an RNAi mechanism and not viainduction of innate immunity.

In vitro and in vivo RACE Analysis of ALN-RSV01 cleavage product. TheRISC-mediated cleavage of a specific mRNA transcript occurs exactly 10nucleotides from the 5′-end of the siRNA antisense strand. Todefinitively confirm an RNAi-mediated mechanism of action for ALN-RSV01,a 5′ Rapid Amplification of cDNA Ends (RACE) assay was used. This assayallows the potential capture and sequence analysis of the specific RNAicleavage product mRNA intermediate following ALN-RSV01 treatment both invitro and in vivo. Following siRNA transfection (200 nM) into Vero cellsand subsequent infection with RSV/A2, a specific cleavage fragment couldbe detected only in the samples treated with ALN-RSV01 as compared toeither PBS or a non-specific siRNA (AL-DP-2153) control (data notshown). In these experiments, 92% of the sequenced clones resulted fromsite-specific cleavage (between positions 26/27 of RSV/A2 N mRNA) (datanot shown). When analyzed in vivo, 60-82% of clones isolated from lungtissue of ALN-RSV01 treated, RSV-infected mice demonstratedsite-specific cleavage of the N-gene transcript between positions 26/27(FIG. 25). Only animals treated with ALN-RSV01, in contradistinctionwith those treated with PBS or mismatch controls, yielded significantnumbers of clones whose sequence was confirmed as the predicted cleavagesite (FIG. 25).

Discussion

RSV can be classified into two subgroups, serotype A and B, which differin all 11 genes identified. Genotypes can vary from year to year withcertain serotypes dominating in some years, but co-circulation ofmultiple RSV strains is commonly observed. Although RSV-A may producegreater viral loads in infants, and RSV-A may produce slightly moresevere disease than RSV-B, both RSV-A and RSV-B produce a similar andclinically indistinguishable disease spectrum. In the case of an RNAitherapeutic, broad spectrum activity is achieved by selecting an siRNAmolecule whose corresponding mRNA target site is conserved across thevarious circulating RSV viral isolates. This is achieved for ALN-RSV01by targeting the relatively conserved viral nucleocapsid which is lessprone to mutation than surface viral proteins such as the G or F.Indeed, within RSV, the N gene is among the most conserved, withapproximately 86% identity at the nucleic acid level and 96% identity atthe amino acid level. Sequence differences within the target site wereobserved at low frequency in clinical specimens, and largely created aG:U wobble between the antisense strand of the siRNA and the target RSVmRNA. All clinical isolates tested were effectively inhibited in vitroby ALN-RSV01 (FIG. 27). Though the IC 50 for the two G:U wobble isolateswas 5 nM (the top of the range), it should be noted that other isolates,namely RUG0420 and MOT0472, both of which show 100% sequenceconservation at the ALN-RSV01 target site, also exhibited 5 nM IC50values. Thus, target site divergence in the non conserved isolates doesnot account for the slightly higher IC50. It is likely that the growthand replication properties of these isolates account for their slightlyhigher IC50 values. These primary isolates spanned genotypes A and Bindicating that ALN-RSV01 is likely active against all currentlycirculating strains. In a large series of in vivo studies, ALN-RSV01 wasfound to be a potent anti-viral in both prophylaxis and treatmentparadigms, showing up to 3 log viral reduction as compared to either PBSor non-specific siRNA controls. Bitko et al. and Zhang et al., have alsopreviously demonstrated siRNA inhibition of RSV in BALB/c mice,targeting either the phosphoprotein (P) mRNA or the non-structural one(NS-1) mRNA, respectively, with reductions similar to those describedhere. While these other siRNAs were effective inhibitors of viralreplication in vivo, neither is ideally suited for RNAi therapeuticdevelopment. The P gene targeting siRNA of Bitko is directed against aregion of the virus that is not conserved across RSV A and B serotypes.Further, the NS-1 targeting siRNA of Zhang et al, is believed tofunction (at least in part), as an immune modulator, by inhibiting theproduction of the RSV protein that inhibits IFN induction.

Applications of ALN-RSV01 in clinical settings will require optimizationof the dosing strategy in terms of dose level and frequency. In thestudy described in this Example, multi-dosing with low doses of siRNAwas equivalent or slightly more effective when compared to theequivalent total dose delivered in a single administration. Therefore,in certain circumstances, multi-dosing may be a preferred strategy fordelivering ALN-RSV01.

Example 17 A Randomized, Double-Blind, Placebo-Controlled Study of anRNAi Therapeutic Directed Against Respiratory Syncytial Virus

This Example describes the significant antiviral activity of ALN-RSV01in adult humans infected with wild-type RSV. Eighty-eight healthysubjects were enrolled into a randomized, double-blind,placebo-controlled trial. A nasal spray of ALN-RSV01 or saline placebowas administered daily for two days before, and for three days after RSVinoculation. RSV was measured serially in nasal washes using multipledifferent viral assays. The results described below show that intranasalALN-RSV01 was well-tolerated, exhibiting a safety profile similar tosaline placebo. The proportion of culture defined RSV infections was71.4% and 44.2% in placebo and ALN-RSV01 recipients respectively(P=0.009), representing a 38% decrease in the number of infected and a95% increase in the number of uninfected subjects. The acquisition ofinfection over time was significantly lower in ALN-RSV01 recipients(P=0.007 and P=0.032, viral culture and PCR respectively). Multiplelogistic regression models showed that the ALN-RSV01 antiviral effectwas independent of other factors including pre-existing RSV antibody andintranasal pro-inflammatory cytokine concentrations.

Materials and Methods

Subjects

The study included healthy males age 18-45. Exclusion criteria included:asthma, smoking, fever or symptomatic respiratory infection within theprevious 2 weeks, medications for rhinitis within the previous 7 days,contact with people at risk for severe RSV, steroid use in the pastmonth, and chronic sinusitis. PCR testing on day −2 showed no RSV A-B,Influenza A-B, Parainfluenza 1, 2, 3 or Human Metapneumovirusinfections.

Study Drug

ALN-RSV01 is a synthetic, double-stranded oligonucleotide. Two21-nucleotide strands in a staggered duplex are formed by hybridizationof two partially complementary single strand RNAs with 19 pairednucleotides and an overhang of two thymidine nucleotides at the 3′ end.The antisense strand is complementary to a 19 nucleotide sequence(residues 3-21) of the mRNA encoding the RSV nucleocapsid N protein.ALN-RSV01 is formulated as a sterile phosphate-buffered solution dilutedin normal saline before dosing. ALN-RSV01 (75 or 150 mg) or placebo(sterile normal saline) was administered by nasal spray(Becton-Dickinson Accuspray®), 0.5 mL/naris.

Inoculating Virus (RSV)

RSV-A (Memphis 37 strain), from an infant hospitalized forbronchiolitis, was GMP-manufactured using FDA-approved vero cells. Theisolate was isolated by plaque-purification and then passaged 5 moretimes before inoculating subjects. RSV identity was confirmed by IFA,electron microscopy and N-gene sequencing. It was determined to be freeof adventitial agents and other human pathogens. Memphis 37 was dilutedin 25% sucrose immediately prior to inoculation of subjects byintranasal drops (0.5 mL/naris).

Study Design and Endpoints

The study was a 1:1 randomized, placebo-controlled, double-blind,parallel-group Phase II trial conducted at a single quarantine unit(Retroscreen Virology Ltd, UK). Local regulatory, institutional reviewboard, and Ethics Committee approval was obtained. All subjects providedwritten informed consent.

Subjects were enrolled through 6 sequential cohorts (FIG. 33). In cohort1 (N=8), ALN-RSV01 was dosed at 75 mg/day. In cohorts 2-6 (N=80),ALN-RSV01 was dosed at 150 mg/day. Cohorts 2-6 consisted of 8, 18, 16,24 and 14 subjects respectively. Subjects were admitted to thequarantine unit for 14 days and were dosed with ALN-RSV01 or placebo 32hrs (Day −1) and 8 hrs (Day 0) prior to RSV inoculation (Day 0). Dailydosing continued after RSV inoculation on Days 1, 2 and 3. The RSVinoculum was determined by plaque assay in HEp-2 cells at the exact timeof inoculation of first and last subjects in each cohort. Nasal washes(5 mL normal saline per naris) for viral assays and cytokinemeasurements were obtained daily on the day of admission to thequarantine unit (Day −2) and on Days 2 through 11. For assessment ofupper respiratory signs and symptoms, a physician's daily directedphysical exam (DPE, Days-2 to 11) and a twice-daily subject-reported RSVsymptom score card (Days −1 to 11) were completed (FIG. 28). Mucusweight for each 24-hour period was recorded from Days −1 through 11.Adverse events were recorded through the Day 28 follow-up visit.

The primary efficacy endpoint was the proportion of subjects who wereRSV infected. Infection was pre-specified as two consecutive positiveRSV assays, the first occurring between Days 2 and 8 post-inoculationinclusively. Additional antiviral efficacy measures included viral areaunder the curve (AUC) and peak viral load. Clinical efficacy endpointsexamined included total symptom score, total DPE score, and mucusweight.

RSV and Antibody Assays

Nasal washes were collected into cold RSV stabilization media,transported on ice, and placed onto HEp-2 cell monolayers within 30minutes of collection. Quantitative culture in HEp-2 cell plaque assayswas performed in 12-well plates using triplicate 10-fold dilutions ofnasal wash as previously described¹⁸. RSV quantitative standards (RSV-ALong ATCC VR-26) were run in parallel with each plaque assay. Nasalwashes containing less than the lower limit of quantification (LLOQ)(<1.7 Log PFU/ml) were considered culture negative.

The quantitative real time RT-PCR (qRT-PCR) assay amplifying an N-genesequence distinct from that of ALN-RSV01 was performed as previouslydescribed¹⁹. Duplicate specimens were run in 96 well platesincorporating internal standards of RNA extracted from parallel aliquotscontaining known RSV-A Long quantity as used in the plaque assays.Results are means of duplicates in Log plaque forming unitequivalents/ml (Log PFUe/ml).

Spin-enhanced cultures were performed using confluent HEp-2 cellmonolayers on cover slips within shell vials and were inoculated with200 μl fresh nasal wash and centrifuged at 700×g for 60 minutes.Monolayers were acetone-fixed after 2 days. Cover slips were evaluatedfor RSV by direct fluorescent antibody techniques using RSV-specificmouse monoclonal antibodies (Bartels, Trinity Biotech, Wicklow,Ireland).

Serum RSV-neutralizing antibodies were measured by a HEp-2 cell RSV 50%microneutralization assay as previously described but performed withMemphis 37 strain. To maximize infection, only subjects with a titer of≦7.67 MU were included, representing the lower third of the normaldistribution in healthy adults (data not shown).

Cytokine Assays

Nasal wash aliquots were analyzed at neat, 1:10 and 1:50 dilutions usingPierce Searchlight chemiluminescent multiplexed sandwich ELISA cytokinearrays (Woburn, Mass., USA) quantifying G-CSF (LLOQ 1.82 pg/mL), IFN-α(LLOQ 7.5 pg/mL), IL-1RA (LLOQ 4.4 pg/mL) and TNF-α (LLOQ 6.7 pg/mL)

Statistical Analysis

Statistical methods used International Conference on Harmonisationguidelines. Continuous values below the LLOQ were set at zero.Continuous efficacy variables, were evaluated using a two-sample t-test.Data not normally distributed was analyzed via the Wilcoxon rank-sumtest. Because the outcome of different cohorts may have been affected byunknown factors and differences in RSV inoculum between cohorts, theprimary efficacy endpoint comparing proportions of subjects infected wasevaluated via a two-sided Cochran-Mantel-Haenszel (CMH) test controllingfor cohort. All analyses were performed using SAS® v8.2 or higher forWindows.

Results

Patients

Eighty-eight subjects were enrolled into the study (FIG. 33). All 88were evaluated for safety, and the 85 who received RSV inoculation wereevaluated for efficacy. All subjects who were RSV-inoculated receivedall study drug doses and evaluations. The treatment groups were wellbalanced at baseline (FIG. 29).

Antiviral Effect

A significantly lower proportion of ALN-RSV01 treated subjects wereinfected compared to placebo (FIG. 30). By quantitative culture,ALN-RSV01 was associated with a 38.1% relative reduction in the numberof infected subjects and a 95.1% increase in the number of uninfectedsubjects (P=0.009). This antiviral effect of ALN-RSV01 was consistentacross the other viral assays with a similar magnitude of effectdemonstrated by qRT-PCR and by spin-culture methods. When only thosesubjects receiving 150 mg of study drug were evaluated (cohorts 2-6) astatistically significant treatment effect was also observed (data notshown).

The timing of the antiviral effect was also examined. The medianincubation period in the placebo group was 3.5 days. By bothquantitative culture and qRT-PCR, the acquisition of infection over timewas reduced in the ALN-RSV01 group (P=0.0069 and P=0.0321 respectively(FIG. 34). This reduction was observed early within 3-4 days postinoculation. No rebound in acquisition of infection was observed. If thedata were censored after day 8, statistical significance remained(P<0.05 for both PCR and culture) (data not shown).

The study was not powered to detect statistically significant effects onviral load. However, study results showed consistent trends towardslower viral load in ALN-RSV01 recipients compared to placebo (FIG. 35),including lower viral AUC and peak viral load (FIG. 35, A-B). Mean dailyviral loads (FIG. 35, C-D) were also lower from Days 4-6 (byquantitative culture) or from Days 4-7 (by qRT-PCR) after viralinoculation, with a maximal difference in mean viral load ofapproximately 1 Log PFU/ml. There were no differences in incubationperiod or duration of viral shedding between ALN-RSV01 and placebo andno viral rebound was observed (FIG. 35, C-D).

To determine whether the effect of ALN-RSV01 on RSV infection wasindependent of other variables, multivariate logistic regressionanalyses were performed evaluating the following variables: treatmentassignment (ALN-RSV01 vs. placebo), RSV inoculum, pre-treatment RSVmicroneutralization titer, and intranasal proinflammatory cytokineconcentrations (TNF-α, IFN-α, G-CSF, and IL-1RA). No statisticallysignificant differences in cytokine AUC or peak cytokine concentrationsoccurred between ALN-RSV01 and placebo groups. The G-CSF concentrationsin the ALN-RSV01 group appeared modestly elevated throughout theobservation period compared to the placebo group whereas all othercytokine concentrations showed inconsistent trends (FIG. 36). Cytokineswere examined by constructing different logistic regression models usingpretreatment cytokine concentration (Day −2), cytokine AUC on Days 2-4following RSV inoculation, and cytokine AUC on Days 2-8 for eachcytokine respectively. Representative models are shown in the table ofFIG. 31. In all models, including those for G-CSF, ALN-RSV01 wasindependently associated with reduced infection (by quantitative cultureor qRT-PCR, P<0.05).

Clinical Effects

Experimental RSV infection of healthy volunteers produces only mild tomoderate upper respiratory tract illness. Within this narrow diseaseseverity spectrum, evaluation of disease measures in subjects treatedwith either ALN-RSV01 or placebo showed no statistically significantdifferences (FIG. 35 E-G). Analysis of mean symptom scores over time inall subjects or in those who were RSV-infected showed that the mostmarked lowering of symptom scores in ALN-RSV01 relative to placebooccurred on Days 4-7 (FIG. 35 H-I). This corresponded to the same timeperiod when there was the greatest reduction in mean daily viral load(FIG. 35 C-D).

Safety

Intranasal ALN-RSV01 was well-tolerated. Adverse events were wellbalanced between ALN-RSV01 and placebo (FIG. 32), with few moderateadverse events (all of which occurred after discharge from quarantine)and no severe or serious adverse events.

In summary, in this randomized, double-blind, placebo-controlled trial,a statistically significant antiviral effect was demonstrated with theRNAi therapeutic, ALN-RSV01. The proportion of subjects infected asdiagnosed by two different culture-based assays was significantly lowerin recipients of ALN-RSV01 as compared to placebo (P<0.05) (FIG. 30).Furthermore, acquisition of infection over time as diagnosed by eitherculture or PCR was also significantly reduced (P<0.01 and P<0.05respectively) (FIG. 34). Finally, multivariate logistical regressionshowed that this ALN-RSV01-associated antiviral effect is statisticallyindependent of the possible effects of other variables (FIG. 31).ALN-RSV01 therefore produces a demonstrable antiviral effect.

Substantial evidence shows that RNAi is the mechanistic basis for theALN-RSV01 antiviral effect. First, ALN-RSV01 reduces RSV in a murinemodel in which mismatched siRNAs (differing from ALN-RSV01 in as few as4 nucleotides) produce no anti-RSV effect. Second, the cleavage productsresulting from RNAi-mediated silencing of the RSV N-protein transcripthave been observed in the lungs of RSV-infected mice treated withALN-RSV01. Third, immunosilent siRNAs targeting the RSV N gene areactive against RSV in mice whereas mismatched siRNAs with potent invitro immunostimulatory activity do not demonstrate anti-RSV activity inmice (R. Meyers, unpublished data). And lastly, substantial evidencewithin this trial itself indicates that ALN-RSV01 is contributing to theobserved antiviral effect independent of intranasal cytokine induction.Multivariate logistic regression models show that the statisticallysignificant ALN-RSV01 antiviral effect was independent of the othervariables examined, including levels of intranasal cytokines. Indeed,where a cytokine effect was evident, there was an association withincreased rather than decreased infection (FIG. 31), likely due to RSVinfection itself stimulating cytokines within human respiratorysecretions. Thus, an antiviral effect which strongly appears mediatedthrough RNAi has been demonstrated.

The timing of ALN-RSV01 can be further optimized to achieve maximalreductions in viral load or RSV disease measures if infection did occur.Here, ALN-RSV01 was dosed twice prior to RSV inoculation and three timessubsequently. ALN-RSV01 was stopped 2-4 days prior to the occurrence ofpeak viral load and peak clinical symptoms of infection. The incubationperiod of RSV in the placebo arm was a median of 3.5 days; thereafter, aconsistently lower daily viral load was found in ALN-RSV01 recipients,and the antiviral effect appeared to continue through Days 6-7post-inoculation with a maximum 1 Log reduction in viral load comparedto placebo. Concurrent to this transient reduction in viral load, themost marked lowering of symptom scores for the ALN-RSV01 group relativeto placebo occurred on Days 4-7. In children, lower RSV loads areassociated with decreased requirements for intensive care, decreasedrespiratory failure and shorter hospitalizations. Therefore, robustreductions in RSV load, if achieved, are likely to translate intoreduced disease severity and clinical benefit. Optimal dosing ofALN-RSV01, so as to be administered through times of active viralreplication, may maximize its effect on viral dynamics and clinicaloutcomes.

There is a time delay in natural RSV infection: RSV infects and causessymptoms in the upper respiratory tract before subsequently moving toinvolve the lungs. In light of the significant effect of intranasalALN-RSV01 shown here on preventing RSV infection when administered priorto and early during the course of infection, aerosolized ALN-RSV01 canbe utilized to alter the spread of an early RSV infection into the lowerairways. The delivery of aerosolized drug simultaneously to both theupper and lower respiratory tracts at varying time intervals followinginfection initiated by relatively low inoculum natural RSV exposures canassist in the optimization of anti-RSV therapy.

Example 18 ALN-RSV01 Specificity in Humans as Analyzed by 5′ RACE Assay

This Example confirms that the antiviral effect of ALN-RSV01 in humansis mediated by RNAi. The 5′ RACE assay is discussed above in more detail(e.g., FIG. 25 and accompanying description).

RNA isolation: Total RNA was purified from nasal samples using RNASTAT-50 LS reagent (Tel-Test) according to manufacturer's instructions.The 5′ RACE assay was done using a modified GeneRacer kit (Invitrogencat#L1502-01). To detect cleavage product, 3 rounds of consecutive PCRwere performed using primers complementary to the RNA adaptor and RSV A2N gene mRNA (GR5′ and Rev for the 1st PCR round; GRN5′ and RevN—for thenested PCR and enrichment PCR).

Name Sequence 5′-3′ SEQ ID NO: GR 5′ CGACTGGAGCACGAGGACACTGA 295 GRN 5′GGACACTGACATGGACTGAAGGAGTA 297 Rev. CCA CTC CAT TTG CTT TTA CAT 296 GATATC C RevN GCT TTT ACA TGA TAT CCC GCA 298 TCT CTG AG

Amplified products were resolved by agarose gel electrophoresis (2.3%agarose) and visualized by ethidium bromide staining. The identity ofspecific cleavage products was confirmed by cloning of the PCR productand sequencing of individual clones.

The results are depicted in FIG. 37. FIG. 37A shows that treatedpatients have a significantly increased frequency of the fragmentpredicted by specific ALN-RSV01 iRNA-mediated cleavage. Moreover, anincrease in specific cleavage correlates strongly with a decrease inobserved viral titer, providing a further indication that the antiviraleffect is mediated by RNAi (FIG. 37B). FIG. 37B also shows that the % ofspecific cleavage observed decreases over time post-dosage. Thus,patient analysis at times proximal to drug dosing may be advantageouswhen measuring this particular marker of drug efficacy.

Example 19 A Multi-Center, Randomized, Double-Blind, Placebo-ControlledStudy of the Safety and Antiviral Activity of Aerosolized ALN-RSV01 PlusStandard of Care in Lung Transplant Recipients Infected with RSV

This prophetic example describes embodiments of the invention relatingto the treatment of patients who have received lung transplants. Theskilled artisan will recognize that many aspects of the methodsdescribed below are equally applicable to treating a patient who has notreceived a lung transplant but is infected with RSV or at risk of RSVinfection.

A study is conducted to assess the safety, tolerability, and antiviralactivity of aerosolized ALN-RSV01 versus placebo, administered in amultiple-dose schedule (once daily for 3 days) to lung transplantrecipients infected with respiratory syncytial virus (RSV). Otherobjectives include evaluating the effects of ALN-RSV01 versus placebo onthe clinical endpoints of RSV infection in lung transplant patients;determining RSV infection characteristics by quantitative RT-PCR(qRT-PCR) analysis of nasal swab and sputum samples, including peakviral load, time to peak viral load, duration of viral shedding, andviral area under the concentration time curve (AUC); and characterizingplasma pharmacokinetics of aerosolized ALN-RSV01.

Study design: randomized, placebo-controlled, double-blind study isundertaken to assess the safety and efficacy of intranasal ALN-RSV01administered to lung transplant recipients infected with RSV. A total of3 doses of ALN-RSV01 are administered to each subject. Up to 21 lungtransplant patients with signs (e.g., decrease in FEV1 and/or fever) andsymptoms of respiratory infection (e.g., new onset rhinorrhea, sorethroat, nasal congestion, cough, wheezing, headache, myalgia, chills,and/or shortness of breath) will be instructed to present to adetermined site for screening as soon as possible (e.g., within 0 to 5days) after identification of the first symptoms.

Patients will have RSV infection confirmed, be randomized to atreatment, and have administration of study medication initiated as soonas possible, e.g., not more than a total of 7 days after onset of signsand symptoms of RSV infection.

Consented eligible patients with positive RSV results will be randomized(2:1) to receive either ALN-RSV01 or placebo (sterile normal saline 0.9%for respiratory administration (saline)) by inhalation once daily for 3days (Study Days 0, 1, and 2). In addition, all patients will receivethe standard of care (SOC) for RSV infection per institutionalguidelines or study investigator practices which may include inpatienthospitalization or out-patient care.

Patients with confirmed RSV infection who are randomized to treatmentwill have nasal swab and sputum samples for RSV determination byquantitative (q)RT-PCR collected daily for 7 days (Day 0 up to Day 6),followed by every other day up to Day 14 days (Day 8, 10, 12, and 14).

Daily outpatient safety assessment visits and respiratory samplecollection (nasal swab and sputum) may take place at the institution orat the subject's home under the care of a visiting nurse trained in thestudy procedures. Assessment of safety may occur up to and include theDay 30 visit (±3 days).

During the Day 90 (±7 days) visit, the following events will beassessed: survival, acute lung rejection, Bronchiolitis obliterans(BOS), intubation, incidence of other viral, bacterial, or fungalrespiratory infections FEV1. Total time on study will be 93 days±7 days

Dosage, route of administration and duration of treatment ofinvestigational drug and control: Patients randomized to ALN-RSV01 willreceive aerosolized ALN-RSV01 (0.6 mg/kg) via a nebulizer, such as aPARI eFlow™ 30L nebulizer, and administered by inhalation once daily for3 days (Days 0, 1, and 2). All doses of ALN-RSV01 will be administeredat the study center by trained study personnel, and the first dose willbe administered with an Investigator on site. The interval between dosesof study medication should ideally be ˜24 hours, but should not be lessthan 12 hours and not more than 36 hours.

For patients receiving bronchodilator therapy, an attempt should be madeto administer study medication within 1 hour after administration ofbronchodilator therapy.

For patients receiving SOC including ribavirin (either by inhalationthree times per day (TID), inhalation overnight for 12-16 hours, or byintravenous (IV) administration), study medication should be given 1 to2 hours prior to the first administration of ribavirin. Subsequent dosesof study medication should be given at the same time of day as thefirst, 1-2 hours prior to the administration of ribavirin with anapproximate 24 hour (12-36 hours) dosing interval between doses of studymedication.

For placebo, randomized patients will receive aerosolized sterile normalsaline via PARI eFlow® 30L nebulizer and administered by inhalation oncedaily for 3 consecutive days. Schedule and timing of placebo with regardto ribavirin and bronchodilators should be the same as above.

Study Assessments and Endpoints: Demographic Data and Medical History:Demographic data, lung transplant related information, and a completemedical history will be obtained at Screen (Day −2 to Day 0).

Physical Examinations: A complete physical examination (PE) will beperformed at Screen (Day −2 to Day 0), on Day 14, and during thefollow-up visit on Day 30. A Directed Physical Examination (DPE) will beconducted at Screen (Day −2 to Day 0) and prior to administration ofstudy medication on Days 0, Day 1, and Day 2; daily on Days 3 to 6, onDay 14, and during the follow-up visits on Day 30. DPE will be performeddaily for the duration of the inpatient period for patients hospitalizedbeyond Day 6. The DPE will focus on the evaluation ofrespiratory-related and infection-related symptoms, using the DPEworksheet will include review of the following body systems: Head, Eyes,Ears, Nose and Throat, Respiratory System, Cardiovascular System, andVital Signs.

Vital signs (blood pressure, pulse rate, oral body temperature, andrespiratory rate) will be measured at Screen (Day −2 to Day 0); Days 0,1 and 2 predose and 1 hour post dose; Days 3 to 6, Days 8-14, and duringthe follow-up visit on Day 30. Vital signs will be assessed daily forthe duration of the inpatient period for patients hospitalized beyondDay 6. Blood pressure (systolic and diastolic), pulse rate, andrespiratory rate will be obtained. Preferably, each patient's bloodpressure will be taken using the same arm throughout the study. Pulserate will be counted for 20 seconds to one minute and recorded in beatsper minute (bpm). Respirations will be recorded in breaths per minute.Body temperature will also be measured.

Height and Weight: Weight (kilograms) will be obtained during thecomplete physical examination at Screen (Day −2 to Day 0) and Day 14,and during the follow up visit on Day 30. Height (centimeters) will beobtained only at Screen (Day −2 to Day 0).

Treatment-Emergent Adverse Events: Treatment-emergent adverse eventswill be evaluated daily from Day 0 (post-dose) through Day 30. After theDay 30 visit, newly reported changes in FEV1 (Volume of air expiredduring the first second of the FVC (Volume expired when going fromcomplete inhalation to complete exhalation as hard and fast as possible)maneuver) or changes in Bronchiolitis obliterans (BOS) classificationwill be documented in the patient Case Report Form (CRF) but will not beconsidered adverse events.

Concomitant Medications: Use of concomitant immunosuppressant,respiratory, and antimicrobial medications will be recorded in thesource documents and on the patient's CRF. Any changes in prior orconcomitant medications during the study will also be recorded on theCRF.

Electrocardiogram: A standard 12 lead Electrocardiogram (ECG) will beobtained at Screen (Day −2 to Day 0) and at Day 14. The PI or designeeis responsible for reviewing the ECG to assess whether the ECG is withinnormal limits and to determine the clinical significance of the results.These assessments will be recorded on the CRF. For any clinicallysignificant abnormal results, the PI or Sub investigator must contactthe Medical Monitor to discuss continued participation of the patient inthe study.

Chest X-ray: Chest X-ray: A standard chest x-ray will be obtained atScreen (Day −2 to Day 0).

Spirometry: Spirometry will be performed at Screen (Day −2 to Day 0),Day 0, 1, and 2 (predose and 1 hour post dose,), on Days 3 to 6 and onDays 14, 30 and 90. All spirometry assessments should be performed aftervital signs are measured. Spirometry performed at Screening, Day 30, andDay 90 must be performed on the same machine having the same calibrationfor internal consistency, and should include absolute values as well as% predicted. For patients hospitalized beyond Day 6, spirometry will beperformed daily for the duration of the inpatient period up to Day 14 oruntil discharge, whichever comes first. Spirometry provides an objectivemethod for assessing the mechanical and functional properties of thelungs and chest wall. Spirometry measures include: Lung capacities e.g.,FVC (volume expired when going from complete inhalation to completeexhalation as hard and fast as possible), which provide a measurement ofthe size of the various compartments within the lung; Volume parametersand flow-rates which measure maximal flow within the airways: FVC is thevolume expired when going from complete inhalation to completeexhalation as hard and fast as possible, The FEV1 is the amount expiredduring the first second of the FVC maneuver; FVC and FEV1 will bemeasured according to the American Thoracic Society/European RespiratorySociety guidelines. If a patient's handheld spirometer does not recordeach of the above protocol specified parameters they may still beenrolled. At a minimum, the FEV1 will be recorded in the sourcedocumentation and Case Report Form (CRF).

Pulse Oximetry: A pulse oximetry reading to assess percent of hemoglobinsaturated with oxygen is to be collected at Screen (Day −2 to Day 0),Day 0 to 6, on Day 14, and during the follow-up visit on Day 30. Pulseoximetry will be assessed daily for the duration of the inpatient periodfor patients hospitalized beyond Day 6 or until discharge from thehospital, whichever comes first.

Clinical Laboratory Tests: Blood and urine samples will be obtained forclinical laboratory testing at Screen (Day 2 to Day 0), on Day 0, 1, and2 (predose), once daily on Day 3 and Day 6, and Days 14 and Day 30 andwill be analyzed by Quintiles Central Laboratory, Morrisville, N.C.Instructions for processing samples for clinical laboratory assessmentare provided in the Quintiles Laboratory Manual. Hematology, ClinicalChemistry, and Urinalysis assessments will include the following:Hematology: Red blood cell (RBC) count, white blood cell (WBC) countwith differential (lymphocytes, monocytes, eosinophils, basophils,neutrophils), mean corpuscular volume (MCV), mean corpuscular hemoglobin(MCH), mean corpuscular hemoglobin concentration (MCHC), hemoglobin(Hgb), hematocrit (Hct) and platelet count; Clinical chemistry: Totalbilirubin, alkaline phosphatase, gamma glutamyltransferase, aspartatetransaminase (AST [SGOT]), alanine transaminase (ALT [SGPT]), lactatedehydrogenase (LDH), creatinine, blood urea nitrogen (BUN), totalprotein, glucose, sodium (Na), potassium (K), calcium (Ca), chloride(Cl); Urinalysis: Visual inspection for appearance and color; dipstickfor pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite,urobilinogen, occult blood. In the event of an unexplained clinicallysignificant abnormal laboratory test occurring after study medicationadministration, the test may be repeated and followed up at thediscretion of the principal investigator (PI) or Sub-investigator untilit has returned to the normal range and/or a diagnosis is made toadequately explain the abnormality. Samples for Clinical LaboratoryAssessments should be packaged and shipped to the central laboratory foranalysis according to instructions provided in the Quintiles laboratorymanual.

Pregnancy Test: Urine and/or serum pregnancy tests will be performed atScreen (Day −2 to Day 0). See the Quintiles laboratory manual fordetails.

Other Laboratory Assessments: Blood samples for cytokines/C-reactiveprotein (CRP) will be obtained during Screen (Day −2 to Day 0), and atintervals during the treatment period. Blood sample collection times forcytokines and CRP are as follows: For hospitalized patients, samples areto be obtained on Day 0: predose, 2, 4, 6, and 8 hours afteradministration of Study Drug; Day 1: predose; Day 2: predose, 2, 4, 6,and 8 hours after study medication administration; and Day 3: 24 hoursafter the last dose. For outpatients, samples should be obtained on Day0: predose, 2, 4, and 6 hours after administration of Study Drug; Day 1:predose; Day 2: predose, 2, 4, and 6 hours after study medicationadministration; and Day 3: 24 hours after the last dose. Cytokineanalyses will include the following: CRP (C-reactive protein),interferon (IFN), interleukin (IL) Ira, IL-6, tumor necrosis factor(TNF)-, and granulocyte colony stimulating factor (G-CSF). Samples forthe cytokine/CRP panel should be packaged and shipped to the centrallaboratory for analysis, according to instructions provided in theQuintiles laboratory manual.

Patient Symptom Card: Acute respiratory symptoms will be self-reportedby patients using a Patient Symptom Card. Patient Symptom cards must befilled out twice daily (morning and evening) from Day −2 to Day 14. Asample Patient Symptom Card is presented in Error! Reference source notfound. The Patient Symptom Card was adopted for use in the current studybecause it was developed for scoring acute, nonspecific respiratorysymptoms, in contrast to well-known asthma and chronic obstructivepulmonary disease (COPD) questionnaires which focus on specificrespiratory symptoms over prolonged periods of time. Symptoms to beassessed include runny nose, stuffy nose, sneezing, sore throat,earache, malaise, cough, shortness of breath at rest, headache muscleand or joint aches, chills, wheezing, chest pain during breathing, andblood in sputum; symptoms will be scored on a scale of 0-3, as follows:(O) Symptom not present; (1) Mild symptom; (2) Moderate symptom; and (3)Severe symptom. Symptoms with scores of 1, 2 or 3 will be reported asadverse events (AEs) by the PI.

Acute Rejection/Bronchiolitis Obliterans: Patients will be evaluated forevidence of acute rejection of the transplant and for the presence orchanges in staging of Bronchiolitis Obliterans Syndrome (BOS) throughDay 90.

Occurrence of Other Bacterial, Viral or Fungal Respiratory Infections:Patients will be evaluated for occurrence of diagnosed and documentedbacterial, viral or fungal respiratory infections (other than RSV)through Day 90. Colonization should not be included.

RSV Infection Characteristics: Nasal swabs and sputum samples foranalysis of RSV titer by qRT-PCR (central RSV laboratory) will beobtained at Screen (Day −2 to Day 0), predose on Days 0, 1 and 2, oncedaily on Days 3 to 6, then once daily on Days 8, 10, 12, and 14. Samplesof BAL fluid will be reserved when available and tested for viral titerby qRT-PCR. Characteristics of RSV kinetics to be evaluated from nasalswab samples will include: Duration of viral shedding (Days) from Day −1to Day 14 of study; Peak viral load (Plaque forming unit equivalent(PFUe)/mL); Time to peak viral load (Days); Mean daily viral load(PFUe/mL); and Overall viral load (based on area under theconcentration-time curve [AUC] in PFUe/mL/day). Nasal swabs, sputumsamples, and samples of Bronchioalveolar lavage (BAL) fluid must beprocessed for shipment to the central RSV laboratory; instructions forprocessing and shipping of these samples are provided in Quintileslaboratory manual.

Pharmacokinetics of ALN-RSV01: Pharmacokinetic assessments will includedetermination of ALN-RSV01 concentrations in plasma and calculation ofderived Pharmacokinetic (PK) parameters from hour 0 on Day 0 to 24 hoursafter the last dose of ALN-RSV01. Sampling times will be as follows:predose, 2, 5, 10, 15, and 30 minutes on Day 0; predose on Day 1;predose, 2, 5, 10, 15, and 30 minutes on Day 2; and 24 hours after thelast dose of study medication on Day 3. PK Parameters that will becalculated (data permitting) include: trough plasma concentration[Cpre]; maximum plasma concentration [Cmax]; time to attain maximumplasma concentration [tmax]; apparent terminal elimination half-life[t½]; apparent total body clearance [CL/F]; apparent volume ofdistribution [Vd/F]; and area under the concentration-time curve[AUC0-last].

Safety Analysis: During the study period and Day 30 Follow-up: Vitalsigns, pulse oximetry, spirometry, adverse events, concomitantmedications, clinical laboratory testing, blood sampling forcytokines/CRP, complete and directed physical examination will beperformed. Interim blinded safety reviews will also be performed after 6and 12 patients complete their Day 6 assessments to review safetyaspects of the study.

Efficacy Analysis: Efficacy assessments will include changes in RSVviral kinetics based on centralized quantitative RT-PCR (qRT-PCR)analysis of nasal swabs and sputum, (as well as, where available BALsamples), and patient symptom scores.

For the Day 90 Follow-up: Survival and FEV1 will be recorded. Anyoccurrences of the following events during the study up to the Day 90visit will be recorded: acute lung rejection, BOS, requirement forintubation, other viral, bacterial, or fungal respiratory infections.

Pharmacokinetic assessments will include determination of ALN-RSV01concentrations in plasma and derived PK parameters from hour 0 to 24hours after the last dose of ALN-RSV01: trough plasma concentration[Cpre], maximum plasma concentration [Cmax], time to attain maximumplasma concentration [tmax], apparent terminal elimination half-life[t½], apparent total body clearance [CL/F], apparent volume ofdistribution [Vd/F], and area under the concentration-time curve[AUC0-last].

Results: ALN-RSV01 is shown to be a safe and effective treatment for theprevention or treatment of RSV infection in human lung transplantrecipients.

This application incorporates all cited references, patents, and patentapplications by references in their entirety for all purposes.

What is claimed is:
 1. A method of reducing the risk of Respiratory Syncytial Virus (RSV) associated bronchiolitis obliterans syndrome (BOS) in a human lung transplant recipient, comprising administering to said human lung transplant recipient a composition comprising a therapeutically effective amount of ALN-RSV01.
 2. The method of claim 1, wherein said administration is intranasal or intrapulmonary.
 3. The method of claim 2, wherein said intrapulmonary administration is by inhalation of said composition.
 4. The method of claim 1 wherein said composition is administered as an aerosol.
 5. The method of claim 4, wherein said aerosol is a nasal spray.
 6. The method of claim 4, wherein said aerosol is produced by a nebulizer.
 7. The method of claim 6, wherein said nebulizer is a PARI eFlow® 30L nebulizer.
 8. The method of claim 1 or 7, comprising administering a plurality of doses of said composition.
 9. The method of claim 8, wherein one of said plurality of doses is administered daily.
 10. The method of claim 8, wherein said plurality of doses is two or three doses.
 11. The method of claim 8, wherein said plurality of doses is three doses.
 12. The method of claim 8, wherein said human lung transplant recipient is presently infected with RSV when the first of said plurality of doses is administered.
 13. The method of claim 1 or 7, wherein said administering reduces RSV protein, RSV mRNA, RSV peak viral load, time to peak RSV viral load, duration of RSV viral shedding, RSV viral AUC, or RSV titer in a cell of the respiratory tract of said human lung transplant recipient.
 14. The method of claim 8, wherein said administering of said plurality of doses reduces RSV protein, RSV mRNA, RSV peak viral load, time to peak RSV viral load, duration of RSV viral shedding, RSV viral AUC, or RSV titer in a cell of the respiratory tract of said human lung transplant recipient to at least the same level as an administering of a single dose that equals the dose provided by said plurality of doses.
 15. The method of claim 8, wherein said administering of said plurality of doses is by inhalation and delivers a total dose of between 0.1 and 0.6 mg/kg of anhydrous oligonucleotide to said human lung transplant recipient.
 16. The method of claim 1 or 7, further comprising determining a characteristic of RSV infection, wherein said characteristic is selected from RSV mRNA, RSV peak viral load, time to peak RSV viral load, duration of RSV viral shedding, RSV viral AUC, or RSV titer.
 17. The method of claim 16, wherein said characteristic of RSV infection is determined by quantitative RT-PCR (qRT-PCR) analysis of a nasal swab sample and/or a sputum sample from said human lung transplant recipient.
 18. The method of claim 1 or 7, wherein said human lung transplant recipient is receiving bronchodilator therapy.
 19. The method of claim 18, wherein said human lung transplant recipient is administered said composition within one hour of receiving bronchodilator therapy.
 20. The method of claim 1 or 7, wherein ribavarin is administered to said human lung transplant recipient.
 21. The method of claim 20, wherein said human lung transplant recipient is administered said composition one to two hours before administration of ribavirin.
 22. The method of claim 1 or 7, wherein said administration of said composition to said human lung transplant recipient is started within seven days of onset of symptoms of RSV infection, wherein said symptoms comprise a decrease in FEV1, fever, new onset rhinorrhea, sore throat, nasal congestion, cough, wheezing, headache, myalgia, chills, or shortness of breath.
 23. The method of claim 1 or 7, wherein said administration of said composition improves FEV1 or causes a reduction in one or more symptoms of RSV infection in said human lung transplant recipient relative to a placebo, wherein said symptoms are selected from the group consisting of fever, new onset rhinorrhea, sore throat, nasal congestion, cough, wheezing, headache, myalgia, chills, or shortness of breath.
 24. The method of claim 1 or 7, wherein the method prevents a reduction in lung function for at least 90 days as determined by measurement of a FEV1 in the lung transplant recipient.
 25. A method of reducing the risk of Respiratory Syncytial Virus (RSV) associated bronchiolitis obliterans syndrome (BOS) in a human lung transplant recipient, comprising administering to said human lung transplant recipient a composition comprising a therapeutically effective amount of ALN-RSV01, wherein the composition is administered daily at 0.6 mg/kg as an aerosol produced by a PARI eFlow® 30L nebulizer.
 26. The method of claim 25, wherein said human lung transplant recipient is receiving bronchodilator therapy.
 27. The method of claim 25, wherein ribavarin is administered to said human lung transplant recipient. 